Methods of using krill oil to treat risk factors for cardiovascular, metabolic, and inflammatory disorders

ABSTRACT

This invention discloses methods of using krill oil and compositions comprising krill oil to treat risk factors for metabolic, cardiovascular, and inflammatory disorders. The present invention also relates to methods of using compositions comprising krill oil to modulate biological processes selected from the group consisting of glucose metabolism, lipid biosynthesis, fatty acid metabolism, cholesterol biosynthesis, and the mitochondrial respiratory chain. The present invention further includes pharmaceutical and/or nutraceutical formulations made from krill oil, methods of making such formulations, and methods of administering them to treat risk factors for metabolic, cardiovascular, and inflammatory disorders.

CROSS-REFERENCE TO RELATED APPLICATION

This is a Continuation-in-Part of U.S. application Ser. No. 12/057,775filed Mar. 28, 2008, which claims benefits from Provisional application61/024,072, filed Jan. 28, 2008, which claims benefits from Provisionalapplication 60/983,446, filed Oct. 29, 2007, which claims benefits fromProvisional application 60/975,058, filed Sep. 25, 2007, which claimsbenefits from Provisional application 60/920,483. The entire contents ofthe prior documents are incorporated herein by reference.

This application claims the benefit of U.S. Prov. Appl. 61/181,743,filed May 28, 2009, and is a continuation-in-part of U.S. applicationSer. No. 12/057,775, filed Mar. 28, 2008, which claims the benefit ofU.S. Prov. Appl. 60/920,483, filed Mar. 28, 2007, U.S. Prov. Appl.60/975,058, filed Sep. 25, 2007, U.S. Prov. Appl. 60/983,446, filed Oct.29, 2007, and U.S. Prov. Appl. No. 61/024,072, filed Jan. 28, 2008, allof which are incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

This invention relates generally to methods of using krill oil to treatrisk factors for metabolic, cardiovascular, and inflammatory disorders,including, but not limited to, modulating endocannabinoidconcentrations; reducing ectopic fat; reducing triacylglycerides in theliver and heart; reducing monoacylglyceride lipase activity in thevisceral adipose tissue, liver, and heart; increasing levels of DHA inthe liver; increasing the levels of EPA and DHA in the phospholipidfractions of tissues that exhibit changes in endocannabinoidconcentration; reducing susceptibility to inflammation, modulatingglucose and lipid homeostasis; reducing fatty liver disease (alcoholicand non-alcoholic); reducing MAGL activity in the heart; increasinglevels of plasma ALA/LA; decreasing levels of ALA/LA in the heart;decreasing levels of ARA in the subcutaneous adipose tissue; anddecreasing availability of substrates to decrease the activity of theendocannabinoid system. The present invention also relates to methods ofusing compositions comprising krill oil to modulate biological processesselected from the group consisting of glucose metabolism, lipidbiosynthesis, fatty acid metabolism, cholesterol biosynthesis, and themitochondrial respiratory chain. The present invention further includespharmaceutical and/or nutraceutical formulations made from krill oil,methods of making such formulations, and methods of administering themto treat risk factors for metabolic, cardiovascular, and inflammatorydisorders.

BACKGROUND OF THE INVENTION

Krill is a small crustacean which lives in all the major oceansworldwide. For example, it can be found in the Pacific Ocean (Euphausiapacifica), in the Northern Atlantic (Meganyctiphanes norvegica) and inthe Southern Ocean off the coast of Antarctica (Euphausia superba).Krill is a key species in the ocean as it is the food source for manyanimals such as fish, birds, sharks and whales. Krill can be found inlarge quantities in the ocean and the total biomass of Antarctic krill(Euphausia superba) is estimated to be in the range of 300-500 millionmetric tons. Antarctic krill feeds on phytoplankton during the shortAntarctic summer. During winter, however, its food supply is limited toice algae, bacteria, marine detritus as well as depleting body proteinfor energy. Virtue et al., Mar. Biol. 126, 521-527. For this reason, thenutritional values of krill vary during the season and to some extentannually. Phleger et al., Comp. Biochem. Physiol. 131 B (2002) 733. Inorder to accommodate variations in food supply, krill has developed anefficient enzymatic digestive apparatus resulting in a rapid breakdownof the proteins into amino acids. Ellingsen et al., Biochem. J. (1987)246, 295-305. This autoproteolysis is highly efficient also post mortem,making it a challenge to catch and store the krill in a way thatpreserves the nutritional quality of the krill. Therefore, in order toprevent the degradation of krill the enzymatic activity is eitherreduced by storing the krill at low temperatures or the krill is madeinto a krill meal.

During the krill meal process the krill is cooked so that all the activeenzymes are denatured in order to eliminate all enzymatic activity.Krill is rich in phospholipids which act as emulsifiers. Thus, it ismore difficult to separate water, fat, and proteins using mechanicalseparation methods than it is in a regular fish meal production line. Inaddition, krill becomes solid, gains weight and loses liquid more easilywhen mixed with hot water. Eventually this may lead to a gradual buildup of coagulated krill proteins in the cooker and a non-continuousoperation due to severe clogging problems. In order to alleviate this,hot steam must be added directly into the cooker. This operation isenergy demanding and may also result in a degradation of unstablebioactive components in the krill oil, such as omega-3 fatty acids,phospholipids and astaxanthin. The presence of these compounds makekrill oil an attractive source as a food supplement, a functional foodproduct, and a pharmaceutical for the animal and human applications.

Omega-3 fatty acids have been shown to have potential effect ofpreventing cardiovascular disease, cognitive disorders, joint diseaseand inflammation-related diseases such as rheumatoid arthritis andosteoarthritis. Astaxanthin is a strong antioxidant and may also assistin promoting optimal health.

Published PCT Application No. WO 00/23546 discloses isolation of krilloil from krill using solvent extraction methods. Krill lipids have beenextracted by placing the material in a ketone solvent (e.g., acetone) inorder to extract the lipid soluble fraction. This method involvesseparating the liquid and solid contents and recovering a lipid richfraction from the liquid fraction by evaporation. Further processingsteps include extracting and recovering by evaporation the remainingsoluble lipid fraction from the solid contents by using a solvent suchas ethanol. The compositions produced by these methods are characterizedby containing at least 75 μg/g astaxanthin, preferably 90 μg/gastaxanthin. Another krill lipid extract disclosed contained at least250 μg/g canastaxanthin, preferably 270 μg/g canastaxanthin.

Published PCT Application No. WO 02/102394 discloses methods of treatingand/or preventing cardiovascular disease, rheumatoid arthritis, skincancer, premenstrual syndrome, diabetes, and enhancing transdermaltransport. The methods include administering a krill or marine oil to apatient. The application also describes a test that was carried out toevaluate the effects of krill and/or marine oils on arterioscleroticcoronary artery disease and hyperlipidemia, and resulted in acholesterol decrease of about 15%, a triglyceride decrease of about 15%,an HDL increase of about 8%, an LDL decrease of about 13%, and acholesterol:HDL ratio decrease of about 14%

Published PCT Application No. WO 2007/080515 discloses a marine lipidextract derived from krill. The extract can be used in methods forpreventing or treating thrombosis.

Korean Published Application No. 2006008155 discloses an oralcomposition comprising a mixture of glucosamine and krill oil (providedin a ratio of 2:3) for use in methods of inhibiting osteoarthritis.

U.S. Pat. No. 7,666,447 discloses compositions including krill extractsand conjugated linoleic acid. The compositions are used in methods fortreating an individual having a disease state selected from the groupconsisting of a joint ailment, PMS, Syndrome X, cardiovascular disease,bone disease and diabetes. The methods comprise administering to theindividual a therapeutically effective amount of a composition includingconjugated linoleic acid and a krill extract comprising krill oil.

However, there remains a need in the art for methods of usingcompositions comprising krill oil to treat risk factors for metabolic,cardiovascular, and inflammatory disorders.

SUMMARY OF THE INVENTION

The present invention provides methods of using compositions comprisingkrill oil (KO) to treat risk factors for metabolic, cardiovascular, andinflammatory disorders, including, but not limited to, modulatingendocannabinoid concentrations; reducing ectopic fat; reducingtriacylglycerides in the liver and heart; reducing monoacylglyceridelipase activity in the visceral adipose tissue, liver, and heart;increasing levels of DHA in the liver; increasing the levels of EPA andDHA in the phospholipid fractions of tissues that exhibit changes inendocannabinoid concentration; reducing susceptibility to inflammation,modulating glucose and lipid homeostasis; reducing fatty liver disease(alcoholic and non-alcoholic); reducing MAGL activity in the heart;increasing levels of plasma ALA/LA; decreasing levels of ALA/LA in theheart; decreasing levels of ARA in the subcutaneous adipose tissue; anddecreasing availability of substrates to decrease the activity of theendocannabinoid system. The present invention also provides methods ofusing compositions comprising krill oil to modulate biological processesselected from the group consisting of glucose metabolism, lipidbiosynthesis, fatty acid metabolism, cholesterol biosynthesis, and themitochondrial respiratory chain. The present invention further includespharmaceutical and/or nutraceutical formulations made from thecompositions, methods of making such formulations, and methods ofadministering them to treat risk factors for metabolic, cardiovascular,and inflammatory disorders.

In some embodiments, the present invention provides methods ofadministering compositions comprising krill oil to treat risk factorsfor metabolic, cardiovascular, and inflammatory disorders in a humansubject, where the method includes the step of administeringcompositions containing krill oil. The risk factors that are treated areselected from the group consisting of modulating endocannabinoidconcentrations; reducing ectopic fat; reducing triacylglycerides in theliver and heart; reducing monoacylglyceride lipase activity in thevisceral adipose tissue, liver, and heart; increasing levels of DHA inthe liver; increasing the levels of EPA and DHA in the phospholipidfractions of tissues that exhibit changes in endocannabinoidconcentration; reducing susceptibility to inflammation, modulatingglucose and lipid homeostasis; reducing fatty liver disease (alcoholicand non-alcoholic); reducing MAGL activity in the heart; increasinglevels of plasma ALA/LA; decreasing levels of ALA/LA in the heart;decreasing levels of ARA in the subcutaneous adipose tissue; anddecreasing availability of substrates to decrease the activity of theendocannabinoid system.

In some embodiments, the present invention provides methods ofadministering compositions comprising krill oil to modulate biologicalprocesses in a human subject, where the method includes the step ofadministering compositions containing krill oil. The biologicalprocesses are selected from the group consisting of glucose metabolism,lipid biosynthesis, fatty acid metabolism, cholesterol biosynthesis, andthe mitochondrial respiratory chain. These biological processes may bemodulated by altering the expression of one or more genes, including,but not limited to, reduced or decreased expression of Ppargc1a(peroxisome proliferator-activated receptor gamma coactivator 1a), Hnf4a(hepatocyte nuclear factor 4 alpha), Pck1 (phosphoenolpyruvatecarboxykinase 1), G6 pc (glucose-6-phosphatase, catalytic), Cpt1a(carnitine palmitoyl transferase 1a), Acads (acyl-coenzyme Adehydrogenase, short chain), Acadm (acyl-coenzyme A dehydrogenase,medium chain), Acadl (acyl-coenzyme A dehydrogenase, long chain), Hmgcr(3-hydroxy-3-methylglutaryl-coenzyme A reductase), Pmvk(phosphomevalonate kinase), Sbref2 (sterol regulatory element bindingfactor 2), Ppargc1b (peroxisome proliferator-activated receptor gammacoactivator 1b), and Sod2 (superoxide dismutase 2). These biologicalprocesses may also be affected by enhanced or increased expression ofNADH (nicotinamide adenine dinucleotide) dehydrogenase and subunitsthereof. The biological processes are also affected by factors includingreduced hepatic glucose production, reduced hepatic gluconeogenesis, andreduced hepatic lipid synthesis.

In some embodiments, the present invention provides methods ofdecreasing lipid content in the liver of a human subject, comprising:administering to said subject an effective amount of a krill oilcomposition under conditions such that lipid content in the liver of thesubject is decreased. In some embodiments, the human subject isclinically obese.

In certain embodiments, the present invention provides methodscomprising providing a krill oil composition to a human subject underconditions such that the cardiovascular disease risk factors of thesubject are improved. In some embodiments, the cardiovascular riskfactors are selected from the group consisting of elevated bloodpressure, elevated serum total cholesterol and low-density lipoproteincholesterol (LDL-C), low serum high-density lipoprotein cholesterol(HDL-C), diabetes mellitus, abdominal obesity, elevated serumtriglycerides, small LDL particles, elevated serum homocysteine,elevated serum lipoprotein(a), prothrombotic factors, fatty liver andinflammatory markers. In some embodiments, the human subject isclinically obese.

In certain embodiments, the present invention provides methodscomprising providing a krill oil composition to a human subject underconditions such that cannabinoid receptor signaling is reduced. In someembodiments, inhibition of the endocannabinoid system of the subjectcomprises lowering the levels of arachidonylethanolamide (AEA) and/or2-arachidonyl glycerol (2-AG). In some embodiments, the human subject isclinically obese.

In certain embodiments, the present invention provides methodscomprising providing a krill oil composition to a human subject; andadministering the krill oil composition to the human subject underconditions such that the appetite of the subject is reduced. In someembodiments, the human subject is clinically obese.

In certain embodiments, the present invention provides methodscomprising providing a krill oil composition to a human subject; andadministering the krill oil composition to the human subject underconditions such that fat accumulation in the subject is reduced. In someembodiments, the human subject is clinically obese.

In certain embodiments, the present invention provides uses of a krilloil composition in a human subject for improvement of cardiovasculardisease risk factors, reduction of cannabinoid receptor signaling,reduction of appetite, reduction of fatty heart or reduction of fataccumulation.

In certain embodiments, the present invention provides uses of krill oilfor the preparation of a medicament for improvement of cardiovasculardisease risk factors, reduction in cannabinoid receptor signaling,reduction of appetite, or reduction of fat accumulation.

In certain embodiments, the present invention provides methodscomprising providing a krill oil composition to a human subject; andadministering the krill oil composition to the human subject underconditions such that the reproductive performance is increased. In someembodiments, reproductive performance is improved chance of ovulation infemales. In some embodiments, reproductive performance isspermatogenesis, sperm motility and/or acreosome reaction.

In certain embodiments, the present invention provides methodscomprising providing a krill oil composition to a human subject; andadministering the krill oil composition to the human subject underconditions such the liver and/or kidney functions are improved.

Other novel features and advantages of the present invention will becomeapparent to those skilled in the art upon examination of the followingor upon learning by practice of the invention.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1. ³¹ P NMR analysis of polar lipids in krill oil.

FIG. 2. Blood lipid profiles in Zucker rats fed different forms ofomega-3 fatty acids (TAG=FO, PL1=NKO and PL2=Superba).

FIG. 3. Plasma glucose concentration in Zucker rats fed different formsof omega-3 fatty acids.

FIG. 4. Plasma insulin concentration in Zucker rats fed different formsof omega-3 fatty acids.

FIG. 5. Estimated HOMA-IR values in Zucker rats fed different forms ofomega-3 fatty acids.

FIG. 6. The effect of dietary omega-3 fatty acids on TNF-α production byperitoneal macrophages.

FIG. 7. The effect of dietary omega-3 fatty acids on lipid accumulationin the liver.

FIG. 8. The effect of dietary omega-3 fatty acids on lipid accumulationin the muscle.

FIG. 9. The effect of dietary omega-3 fatty acids on lipid accumulationin the heart.

FIG. 10. Relative concentrations of DHA in the brain in Zucker ratssupplemented with omega-3 fatty acids.

FIG. 11. Mean group body weights (g) in the collagen-induced male DBA/1arthritic mice. B-PL2 is the krill oil group. * p<0.05, significantlydifferent from Group A (Positive Control—Fish Oil) and Group C(Control).

FIG. 12. Body weight for the various treatment groups.

FIG. 13. Muscle weight for the various treatment groups.

FIG. 14. Muscle to body weight ratio for the various treatment groups.

FIG. 15. Serum adiponectin levels (ng/ml) for the various treatmentgroups.

FIG. 16. Serum insulin levels for the various treatment groups.

FIG. 17. Blood glucose (mmol/l) levels in the various treatment groups.

FIG. 18. HOMA-IR values for the various treatment groups.

FIG. 19. Liver triglyceride levels (μmol/g) for the various treatmentgroups.

FIG. 20A-B. Levels of anandamide (arachidonoyl ethanolamide) and2-arachidonoyl glycerol in visceral adipose tissue in Zucker rats.

FIG. 21A-B. Levels of anandamide (arachidonoyl ethanolamide) and2-arachidonoyl glycerol in subcutaneous adipose tissue in Zucker rats.

FIG. 22A-B. Levels of anandamide (arachidonoyl ethanolamide) and2-arachidonoyl glycerol in liver tissue in Zucker rats.

FIG. 23A-B. Levels of anandamide (arachidonoyl ethanolamide) and2-arachidonoyl glycerol in heart tissue in Zucker rats.

FIG. 24. Triacylglyceride content in liver.

FIG. 25. Triacylglyceride content in heart.

FIG. 26. Cholesterol profile in plasma.

FIG. 27. Fatty acid analyses of monocytes.

FIG. 28. TNF alpha release in peritoneal monocytes after ex vivochallenge with LPS.

FIG. 29A-B. Liver (A) and heart (B) triacylglycerol concentrations ofobese Zucker rats fed control, fish oil, or krill oil diets for fourweeks. Values are expressed as mean+/− SD, n=6. Means that do not have acommon letter differ, P<0.05.

FIG. 30A-B. Visceral AEA (A) and 2-AG (B) concentrations in obese Zuckerrats fed control, fish oil, or krill oil diets for four weeks. Valuesare expressed as mean+/− SD, n=6. Means that do not have a common letterdiffer, P<0.05.

FIG. 31A-D. Liver (A and B) and heart (C and D) AEA (A and C) and 2-AG(B and D) concentrations in rats fed control, fish oil, or krill oildiets for four weeks. Values are expressed as mean+/− SD, n=6. Meansthat do not have a common number differ, P<0.05.

FIG. 32A-B. Cholesterol (A) and TAG (B) concentrations in plasma fromrats fed control (C), fish oil (FO), or krill oil (KO) diets. Error barsdepict S.D., n=6. Different letters denote significant differences(p<0.05)

FIG. 33. Treatment-induced changes in the expression of themitochondrial reactive oxygen species detoxification enzyme Sod2.Expression was significantly decreased by a KO diet.

FIG. 34. Genes suggesting decreased glucose uptake and increasedfructose metabolism. KO diet showed a trend for increased Aldobexpression (p=0.022)

FIG. 35. Key genes regulating hepatic glucose production

FIG. 36. Key genes involved in fatty acid metabolism.

FIG. 37. Key genes regulating cholesterol biosynthesis in the liver3-hydroxy-3-methylglutaryl-Coenzyme A

FIG. 38. Transcriptional cofactors and gene targets proposed to mediatethe effect of krill-supplements on hepatic glucose metabolism and lipidbiosynthesis.

DEFINITIONS

An “ether phospholipid” as used herein preferably refers to aphospholipid having an ether bond at position 1 of the glycerolbackbone. Examples of ether phospholipids include, but are not limitedto, alkylacylphosphatidylcholine (AAPC),lyso-alkylacylphosphatidylcholine (LAAPC), andalkylacylphosphatidylethanolamine (AAPE). A “non-ether phospholipid” isa phospholipid that does not have an ether bond at position 1 of theglycerol backbone.

As used herein, the term “omega-3 fatty acid” refers to polyunsaturatedfatty acids that have the final double bond in the hydrocarbon chainbetween the third and fourth carbon atoms from the methyl end of themolecule. Non-limiting examples of omega-3 fatty acids include,5,8,11,14,17-eicosapentaenoic acid (EPA),4,7,10,13,16,19-docosahexaenoic acid (DHA) and7,10,13,16,19-docosapentaenoic acid (DPA).

As used herein, the term “w/w (weight/weight)” refers to the amount of agiven substance in a composition on weight basis. For example, acomposition comprising 50% w/w phospholipids means that the mass of thephospholipids is 50% of the total mass of the composition (i.e., 50grams of phospholipids in 100 grams of the composition, such as an oil).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods of using krill oil and/orcompositions comprising krill oil to treat risk factors for metabolic,cardiovascular, and inflammatory disorders, including, but not limitedto, modulating endocannabinoid concentrations; reducing ectopic fat;reducing triacylglycerides in the liver and heart; reducingmonoacylglyceride lipase activity in the visceral adipose tissue, liver,and heart; increasing levels of DHA in the liver; increasing the levelsof EPA and DHA in the phospholipid fractions of tissues that exhibitchanges in endocannabinoid concentration; reducing susceptibility toinflammation, modulating glucose and lipid homeostasis; reducing fattyliver disease (alcoholic and non-alcoholic); reducing MAGL activity inthe heart; increasing levels of plasma ALA/LA; decreasing levels ofALA/LA in the heart; decreasing levels of ARA in the subcutaneousadipose tissue; and decreasing availability of substrates to decreasethe activity of the endocannabinoid system.

The present invention also relates to method of using krill oil and/orcompositions comprising krill oil to modulate biological processesselected from the group consisting of glucose metabolism, lipidbiosynthesis, fatty acid metabolism, cholesterol biosynthesis, and themitochondrial respiratory chain.

The present invention further includes pharmaceutical and/ornutraceutical formulations made from krill oil, methods of making suchformulations, and methods of administering them to treat risk factorsfor metabolic, cardiovascular, and inflammatory disorders.

A. Methods of Using Krill Oil

The present invention relates to methods of using krill oil andcompositions comprising krill oil to treat one or more risk factors formetabolic, cardiovascular, and inflammatory disorders. There are avariety of risk factors associated with metabolic, cardiovascular,inflammatory, and other disorders, and it has been found in accordancewith the present invention that krill oil significantly modulates asubstantial number of risk factors associated with metabolic,cardiovascular, inflammatory and other disorders. These disorders mayinclude, but are not limited to, obesity, type II diabetes, type Idiabetes, gestational diabetes, metabolic syndrome, dyslipidemia,hypercholesterolemia, hypertension, coronary artery disease,atherosclerosis, stroke, rheumatoid arthritis, and osteoarthritis.

The level(s) of the risk factor(s) to be treated may be assessed in oneor more body fluids of interest, including, but not limited to, blood,plasma, urine, sweat, tears, and cerebrospinal fluid. The level(s) ofthe risk factor(s) may also be assessed in one or more organs ofinterest, including, but not limited to, the brain, heart, liver, bloodvessels, visceral adipose tissue (VAT), subcutaneous adipose tissue(SAT), lungs, intestines, blood vessels, lymph nodes, kidneys, andpancreas.

Specific risk factors that may be modulated by the krill oil-basedcompositions in the methods of the present invention includeendocannabinoid concentrations (particularly AEA(N-arachidonoylethanolamine (anandamide)) and 2-AG(2-arachidonoylglycerol) in the liver, heart, and VAT, although thepresent invention is not limited to these endocannabinoids); ectopicfat; triacylglycerides in the liver and heart; monoacylglyceride lipaseactivity in the VAT, liver, and heart; susceptibility to inflammation,glucose and lipid homeostasis; fatty liver disease (alcoholic andnon-alcoholic); MAGL (monoacylglycerol lipase) activity in the heart;levels of ALA/LA (alpha-linolenic acid/linoleic acid) in the heart;levels of ARA (arachidonic acid) in the SAT; and availability ofsubstrates to decrease the activity of the endocannabinoid system.According to certain aspects of the invention, the levels orconcentrations of these risk factors are decreased in a subjectsuffering from or at risk for a metabolic, cardiovascular, orinflammatory disorder by administering a krill oil composition.According to other aspects of the invention, the levels orconcentrations of these risk factors are decreased in a patientpopulation, by administering a krill oil composition to a patientpopulation including individuals suffering from or at risk for ametabolic, cardiovascular, or inflammatory disorder. According to someaspects of the invention, a krill oil composition may be administered toa subject or patient population in accordance with methods for reducinglevels of one or more of these risk factors relative to the level ofexpression or activity seen in an individual or population not sufferingfrom a metabolic, cardiovascular, inflammatory disorder.

Other risk factors that modulated by the krill-based compositions in themethods of the present invention include levels of DHA (docosahexaenoicacid) in the liver; levels of EPA (eicosapentaenoic acid) and DHA in thephospholipid fractions of tissues that exhibit changes inendocannabinoid concentration; and levels of plasma ALA/LA. According tocertain aspects of the invention, the levels or concentrations of theserisk factors are increased in a subject suffering from or at risk for ametabolic, cardiovascular, or inflammatory disorder by administering akrill oil composition. According to other aspects of the invention, thelevels or concentrations of these risk factors are increased in apatient population, by administering a krill oil composition to apatient population including individuals suffering from or at risk for ametabolic, cardiovascular, or inflammatory disorder. According to someaspects of the invention, a krill oil composition may be administered toa subject or patient population in accordance with methods forincreasing levels of one or more of these risk factors relative to thelevel of expression or activity seen in an individual or population notsuffering from a metabolic, cardiovascular, inflammatory disorder.

Biological processes that may be modulated by krill oil and compositionscontaining krill oil in the methods of the present invention includeglucose metabolism, gluconeogenesis, lipid biosynthesis, fatty acidmetabolism, cholesterol biosynthesis, and the mitochondrial respiratorychain. These biological processes are affected by expression of a numberof genes, including, but not limited to, reduced or decreased expressionof Ppargc1a (peroxisome proliferator-activated receptor gammacoactivator 1a), Hnf4a (hepatocyte nuclear factor 4 alpha), Pck1(phosphoenolpyruvate carboxykinase 1), G6 pc (glucose-6-phosphatase,catalytic), Cpt1a (carnitine palmitoyl transferase 1a), Acads(acyl-coenzyme A dehydrogenase, short chain), Acadm (acyl-coenzyme Adehydrogenase, medium chain), Acadl (acyl-coenzyme A dehydrogenase, longchain), Hmgcr (3-hydroxy-3-methylglutaryl-coenzyme A reductase), Pmvk(phosphomevalonate kinase), Sbref2 (sterol regulatory element bindingfactor 2), Ppargc1b (peroxisome proliferator-activated receptor gammacoactivator 1b), and Sod2 (superoxide dismutase 2). These biologicalprocesses may also be affected by enhanced or increased expression ofNADH (nicotinamide adenine dinucleotide) dehydrogenase and subunitsthereof. The biological processes are also affected by factors includingreduced hepatic glucose production, reduced hepatic gluconeogenesis, andreduced hepatic lipid synthesis.

These risk factors may be modulated by increasing or decreasing (asappropriate) the expression of a gene, activity of an enzyme, etc.,relative to the level of expression or activity seen in an individual orpopulation not suffering from a metabolic, cardiovascular, inflammatory,or other disorder. Alternatively, the various genes, enzymes, and otherrisk factors may be modulated by increasing or decreasing (asappropriate) the expression of a gene, activity of an enzyme, etc.,relative to the level of expression or activity seen in an individual orpopulation suffering from a metabolic, cardiovascular, inflammatory, orother disorder to be treated or prevented.

The risk factors modulated in accordance with the methods of preventingor treating a metabolic, cardiovascular, inflammatory, or other disordermay be modulated to a degree that results in improvement in the symptomsof the disorder, elimination of the disorder, or reduction in risk fordeveloping the disorder. In these aspects, it may be useful to establisha baseline level for the risk factor in a subject or patient populationbeing treated by determining the amount of the risk factor present in abody fluid or organ of interest. Such a baseline could be determined byassessing the amount of one or more risk factors present in a body fluidor tissue sample taken from a subject or patient population, prior toany treatment with krill oil. According to some aspects, the krill oilor krill oil composition is then administered in an amount that issufficient to result in an increase/decrease (as appropriate) in a levelof a risk factor observed in a subject or patient population beingtreated by the methods of the invention. According to further aspects,the increase/decrease is at least 5% relative to the baseline level.Preferably the level of the risk factor is increased/decreased by atleast 10%, at least 20%, at least 35%, at least 50%, at least 65%, atleast 80%, at least 90%, or at least 95%, relative to the baselinelevel. In some embodiments, the level of the risk factor may beincreased/decreased by 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%,900%, 1000% or more as compared to the baseline level by the methods ofthe present invention.

Cardiovascular Disease

In certain embodiments, the present invention can be used in methods ofdecreasing cardiovascular disease risk factors of a subject. In someembodiments, the cardiovascular risk factors are selected from the groupconsisting of elevated blood pressure, elevated serum total cholesteroland low-density lipoprotein cholesterol (LDL-C), low serum high-densitylipoprotein cholesterol (HDL-C), diabetes mellitus, abdominal obesity,elevated serum triglycerides, small LDL particles, elevated serumhomocysteine, elevated serum lipoprotein(a), prothrombotic factors,fatty liver and inflammatory markers. In some embodiments, the subjectis a human, and in other embodiments, the subject is clinically obese.

In some embodiments, the krill oil composition of the present inventionfind use in the treatment of fatty heart disease, alcoholic fatty liverdisease, and non-alcoholic fatty liver disease. Thus, the krill oilcompositions are useful for decreasing the lipid content of the heartand/or liver of a subject. In other embodiments, the present inventionprovides methods of providing a krill oil composition to a subject; andadministering the krill oil composition to the subject under conditionssuch the liver and/or kidney functions are improved. In someembodiments, the subject is a human, and in other embodiments, thesubject is clinically obese.

Obesity

Excess adipose tissue mass (overweight and obesity) is associated withlow grade inflammation in adipose tissue and in the whole bodyreflecting the inflammatory mediators “spilling over” from fat tissue.Inflammation appears to be an important link between obesity andmetabolic syndrome/type-II diabetes as well as cardiovascular disease.Thus, excess adipose tissue is an unhealthy condition.

Weight reduction will improve the inflammatory condition, but persistentweight reduction is difficult to achieve. Omega-3 fatty acidsupplementation may alleviate the inflammatory condition in adiposetissue and thus ideally complement the principal strategies of weightreduction, i.e., low calorie diet and exercise. Although a diet andexercise regime may fail to result in a consistent decrease in weightover the long term, the effect of omega-3 fatty acids alleviating theinflammatory condition in the adipose tissue may persist, generating acondition that can be described as “healthy adipose tissue”. Reductionin adipose tissue inflammation may be achieved by increasing circulatinglevels of adiponectin. Adiponectin is an adipose tissue derivedanti-inflammatory hormone.

This aspect of the invention therefore relates to the discovery thatkrill oil is highly effective in alleviating negative health effectscaused by obesity, such as reducing LDL cholesterol, reducing ectopicfat deposition and reducing susceptibility to inflammation. Thesenegative health effects may lead to increased cardiovascular diseaserisk. Accordingly, another embodiment of the invention is to use krilloil in overweight and obese subjects for alleviating diet-inducedadipose tissue dysfunction and diet induced changes in lipid metabolism.

In certain embodiments, the present invention provides methodscomprising providing a krill oil composition to a subject; andadministering the krill oil composition to the subject under conditionssuch that the appetite of the subject is reduced. In some embodiments,the subject is a human, and in other embodiments, the subject isclinically obese.

In certain embodiments, the present invention provides methodscomprising providing a krill oil composition to a subject; andadministering the krill oil composition to the subject under conditionssuch that fat accumulation in the subject is reduced. In someembodiments, the subject is a human, and in other embodiments, thesubject is clinically obese.

In still other embodiments, the krill oil compositions of the presentinvention find use in increasing or inducing diuresis. In someembodiments, the krill oil compositions of the present invention finduse in decreasing protein catabolism and increasing the muscle mass of asubject.

Type 2 Diabetes and Metabolic Disorder

Type 2 diabetes is a metabolic disorder characterized by impairedglycemic control (high blood glucose levels). In type 2 diabetes,tissue-wide insulin resistance contributes to the development of thedisease. Strategies for reducing insulin resistance or improving tissuesensitivity to insulin are recognized as beneficial in preventing type 2diabetes. In further embodiments, krill oil is effective in reducingrisk factors of type 2 diabetes such as hyperinsulinemia and insulinresistance.

The methods of the present invention may be used to treat and/or preventtype II diabetes and metabolic syndrome in a subject, or reduce theincidence of type II diabetes and/or metabolic syndrome in a patientpopulation comprising individuals at risk for developing diabetesand/metabolic syndrome. Another embodiment of the invention provides akrill oil composition effective for improving the blood lipid profile byincreasing the HDL cholesterol levels, decreasing the LDL cholesteroland triglyceride levels. Hence the novel krill oil composition iseffective for treating metabolic syndrome, which is defined as thecoexistence of 3 or more components selected from the group: abdominalobesity, high serum triglyceride levels, low HDL levels, elevated bloodpressure and high fasting plasma glucose levels. In another embodimentof the invention, krill oil compositions are provided that are effectiveand safe for the treatment of type II diabetes and metabolic syndrome inhumans.

Endocannabinoid Modulation

In certain embodiments, the present invention provides methodscomprising providing a krill oil composition to a subject underconditions such that cannabinoid receptor signaling is reduced. In someembodiments, inhibition of the endocannabinoid system of the subjectcomprises lowering the levels of arachidonylethanolamide (AEA) and/or2-arachidonyl glycerol (2-AG). In some embodiments, the subject is ahuman, and in other embodiments, the subject is clinically obese.

The endocannabinoid system consists of cannabinoid (CB) receptors,endocannabinoids (EC) and enzymes involved in the synthesis anddegradation of these molecules. Cannabionoid-1 (CB-1) receptors arelocated in the central nervous system such as the brain (basal ganglia,limbic system, cerebellum and hippocampus) and the reproductive system(both male and female), but also peripherally in liver, muscle, anddifferent adipose tissues. Cannabionoid-2 (CB-2) receptors are locatedon immune cells and in the spleen.

A dysregulated endocannabinoid system results in excessive eating andfat accumulation and is therefore likely to play an important role inthe pathogenesis of obesity. This chronic activation may not only becaused by obesity, but also by high fat diets, which can predispose thebody to enhanced endocannabinoid biosynthesis.

The present invention discloses that krill oil can be used toeffectively modulate the endocannabinoid system in Zucker fatty rats. Itis shown that krill oil is more effective than fish oil and the controldiet in reducing the level of endocannabinoids AEA and 2-AG in visceraladipose in this model. Visceral fat is the metabolically more active fatand accumulation of visceral fat has been associated with insulinresistance, glucose intolerance, dyslipidemia, hypertension and coronaryheart disease. Accumulation of visceral fat is initiated where thecapacity for storing subcutaneous fat is saturated. The Zucker rats areleptin receptor-deficient animals, and therefore they became obese dueto the increased feed intake which gradually results in the developmentof metabolic syndrome (the rats develop hyperglycemia, ectopic fatdeposition, and elevated LDL cholesterol levels). The data show that thereduction in 2-AG levels in subcutaneous adipose is the most pronouncedwhile the level of AEA in liver and heart were also clearly reducedafter intake of krill oil compared to all the other treatments.Subcutaneous fat is less metabolic active than visceral adipose tissue.Functional effects of a dysregulated endocannabinoid system wereobserved in Example 12, as the rats developed fatty heart, fatty liver,hyperglycemia and elevated LDL cholesterol levels.

Krill oil is an effective agent for modulating the endocannabinoidsystem, and thereby alleviating the negative health effects of obesity.The invention also relates to the discovery that krill oil is effectivein reducing the level of the endocannabinoid precursors, i.e., thearachidonic acid content in phospholipids in the heart, subcutaneousadipose tissue, and visceral adipose tissue. The TAG fraction of thevisceral and subcutaneous adipose tissue was influenced by omega-3supplementation, showing an increased incorporation of EPA (30 fold),DHA (10 fold) and DPA (10 fold). However, the large increase in TAG isless metabolically important than the small increase in thephospholipids. The AEA and 2-AG concentration in visceral and adiposetissue mirrors the fatty acid profiles. Liver TAG omega-3 weresignificantly increased in fish oil and krill oil groups whereas nochanges were found in arachidonic acid or other omega-6 fatty acids.Heart TAGs fatty acids showed increased levels of EPA, DPA and DHA anddecrease in ARA in the phospholipid fraction.

The various methods of the present invention demonstrate that krill oilis effective in changing endocannabinoid receptor signaling bymodulating the level of the cannabinoid receptor ligands. The levels ofendocannabinoid precursors, i.e., arachidonic acid attached tophospholipids, are reduced as well. It might be that the high level ofomega-3 phospholipids play a role in the effective modulation of theendocannabinoid system, however the mechanism of action by which krilloil works remains unknown at this stage.

The present invention also relates to modulation of the endocannabinoidsystem in tissues such as kidney, testis, different brain areas,intestines, pancreas, thyroids glands, etc. A preferred embodiment ofthis invention is the use of krill oil for modulation of a dysfunctionalendocannabinoid system in all tissues in order to obtain improvedhealth. Non-limiting examples of such health effects are treatment ofobesity, reduction in feed intake, increased energy expenditure,reduction in cholesterol, improvement in male reproduction(spermatogenesis, sperm motility and acreosome reaction) and femalereproduction (increased ovulation), increased sexual drive (libido),treatment of atherosclerosis, improvement in bone metabolism,improvement in lipid metabolism, treatment of ectopic fat deposition,treatment of liver disease such as fibrosis and cirrhosis, control ofglucose homeostasis, improvement in insulin resistance, treatment offatty heart and cardiomyopathy.

B. Krill Oil

According to some aspects, the various methods of the present inventionmay be carried out using krill oil, or compositions comprising krilloil. The krill oil is characterized by containing high levels ofastaxanthin, phospholipids, included an enriched quantities of etherphospholipids, and omega-3 fatty acids.

Krill oil is obtained from Antarctic krill (Euphausia Superba) byextracting the lipids with supercritical and/or liquid solvents. Krilloil is different from fish oil at least in the respect that it containsastaxanthin, and the majority of the omega-3 fatty acids are attached tophospholipids.

In preferred embodiments, the krill oil compositions are made asdescribed in co-pending application PCT/GB2008/001080, which isincorporated herein by reference. In other preferred embodiments,compositions for use in accordance with the invention may include, butare not limited to, Superba™ krill oil (Aker Biomarine, Norway). Inother preferred embodiments, the compositions for use in accordance withthe invention comprise krill oil that is obtained from krill meal byethanol extraction and/or CO₂ extraction. However, any suitable methodsfor extracting oil from krill may be used in accordance with the presentinvention.

The krill oil-containing compositions that are preferably used in orderto carry out the methods of the present invention are distinguished frompreviously-described krill oil products, such as those described in U.S.Pat. No. 6,800,299 or WO 03/011873 and Neptune brand krill oil (NKO®,Neptune Technologies & Bioressources, Laval, Quebec, Canada), by havingsubstantially higher levels of non-ether phospholipids, etherphospholipids, and astaxanthin.

The krill compositions that may be used in accordance with the presentinvention are preferably derived from Euphausia superba. Regardless ofthe krill used, the compositions preferably comprise from about 40% toabout 60% w/w phospholipids, preferably from about 45% to 55% w/wphospholipids and from about 300 mg/kg astaxanthin to about 2500 mg/kgastaxanthin, preferably from about 1000 to about 2200 mg/kg astaxanthin,more preferably from about 1500 to about 2200 mg/kg astaxanthin. In somepreferred embodiments, the compositions comprise greater than about1000, 1500, 1800, 1900, 2000, or 2100 mg/kg astaxanthin.

In some preferred embodiments, the krill compositions of the presentinvention comprise from about 1%, 2%, 3% or 4% to about 8%, 10%, 12% or15% w/w ether phospholipids or greater than about 4%, 5% 6%, 7%, 8%, 9%or 10% ether phospholipids. In some embodiments the ether phospholipidsare preferably alkylacylphosphatidylcholine,lyso-alkylacylphosphatidylcholine, alkylacylphosphatidyl-ethanolamine orcombinations thereof. In some embodiments, the krill compositionscomprise from about 1%, 2%, 3% or 4% to about 8%, 10%, 12% or 15% w/wether phospholipids and from about 30%, 33%, 40%, 42%, 45%, 48%, 50%,52%, 54%, 55% 56%, 58% to about 60% non-ether phospholipids so that thetotal amount of phospholipids (both ether and non-ether phospholipids)ranges from about 40% to about 60%. One of skill in the art willrecognize that the range of 40% to 60% total phospholipids, as well asthe other ranges of ether and non-ether phospholipids, can include othervalues not specifically listed within the range.

In further embodiments, the compositions comprise from about 20% to 45%w/w triglycerides; and from about 400 to about 2500 mg/kg astaxanthin.In some embodiments, the compositions comprise from about 20% to 35%,preferably from about 25% to 35%, omega-3 fatty acids as a percentage oftotal fatty acids in the composition, wherein from about 70% to 95%, orpreferably from about 80% to 90% of the omega-3 fatty acids are attachedto the phospholipids.

The krill oil extracted for use in the methods of the present inventioncontains few enzymatic breakdown products. Examples of the krill oilcompositions of the present invention are provided in Tables 4-19. Insome embodiments, the present invention provides a polar krill oilcomprising at least 65% (w/w) of phospholipids, wherein thephospholipids are characterized in containing at least 35% omega-3 fattyacid residues. The present invention is not limited to the presence ofany particular omega-3 fatty acid residues in the krill oil composition.In some preferred embodiments, the krill oil comprises EPA and DHAresidues. In some embodiments, the krill oil compositions comprise lessthan about 5%, 4%, 3% or preferably 2% free fatty acids on aweight/weight (w/w) basis. In some embodiments, the krill oilcompositions comprise less than about 25%, 20%, 15%, 10% or 5%triglycerides (w/w). In some embodiments, the krill oil compositionscomprise greater than about 30%, 40%, 45%, 50%, 55%, 60%, or 65%phosphatidyl choline (w/w). In some embodiments, the krill oilcompositions comprise greater than about 100, 200, 300, 400, or 500mg/kg astaxanthin esters and up to about 700 mg/kg astaxanthin esters.In some embodiments, the present invention provides krill oilcompositions comprising at least 500, 1000, 1500, 2000, 2100, or 2200mg/kg astaxanthin esters and at least 36% (w/w) omega-3 fatty acids. Insome embodiments, the krill oil compositions of the present inventioncomprise less than about 1.0 g/100 g, 0.5 g/100 g, 0.2 g/100 g or 0.1g/100 g total cholesterol. In some embodiments, the krill oilcompositions of the present invention comprise less than about 0.45.

In some embodiments, the present invention is carried out using aneutral krill oil extract comprising greater than about 70%, 75% 80%,85% or 90% triglycerides. In some embodiments, the krill oilcompositions comprise from about 50 to about 2500 mg/kg astaxanthinesters. In some embodiments, the krill oil compositions comprise fromabout 50, 100, 200, or 500 to about 750, 1000, 1500 or 2500 mg/kgastaxanthin esters. In some embodiments, the compositions comprise fromabout 1% to about 30% omega-3 fatty acid residues, and preferably fromabout 5%-15% omega-3 fatty acid residues. In some embodiments, the krilloil compositions comprise less than about 20%, 15%, 10% or 5%phospholipids.

In some embodiments, the present invention is carried out using krilloil containing less than about 70, 60, 50, 40, 30, 20, 10, 5 or 1micrograms/kilogram (w/w) astaxanthin esters. In some embodiments, thekrill oil is clear or only has a pale red color. In some embodiments,the low-astaxanthin krill oil is obtained by first extracting a krillmaterial, such as krill oil, by supercritical fluid extraction with neatcarbon dioxide. It is contemplated that this step removes astaxanthinfrom the krill material. In some embodiments, the krill material is thensubjected to supercritical fluid extraction with carbon dioxide and apolar entrainer such as ethanol, preferably about 20% ethanol. The oilextracted during this step is characterized in containing low amounts ofastaxanthin. In other embodiments, krill oil comprising astaxanthin isextracted by countercurrent supercritical fluid extraction with neatcarbon dioxide to provide a low-astaxanthin krill oil.

In some embodiments, the present invention is carried out using krilloil that is substantially odorless. By substantially odorless it ismeant that the krill oil lacks an appreciable odor as determined by atest panel. In some embodiments, the substantially odorless krill oilcomprises less than about 10, 5 or 1 milligrams/kilogram trimethylamine.In some preferred embodiments, the odorless krill oil is produced byfirst subjecting krill material to supercritical fluid extraction withneat carbon dioxide to remove odor causing compounds such astrimethylamine, followed by extraction with carbon dioxide with a polarentrainer such as ethanol.

C. Krill Oil-Based Compositions

In some embodiments, the present invention provides encapsulatedEuphausia superba krill oil compositions. In some embodiments, thepresent invention provides a method of making a Euphausia superba krilloil composition comprising contacting Euphausia superba with a polarsolvent to provide an polar extract comprising phospholipids, contactingEuphausia superba with a neutral solvent to provide a neutral extractcomprising triglycerides and astaxanthin, and combining said polarextract and said neutral extract to provide the Euphausia superba krilloils described above.

In some embodiments, fractions from polar and non-polar extractions arecombined to provide a final product comprising the desired etherphospholipids, non-ether phospholipids, omega-3 moieties andastaxanthin. In other embodiments, the present invention providesmethods of making a Euphausia superba (or other krill species) krill oilcomprising contacting a Euphausia superba preparation such as Euphausiasuperba krill meal under supercritical conditions with CO₂ containing alow amount of a polar solvent such as ethanol to extract neutral lipidsand astaxanthin; contacting meal remaining from the first extractionstep under supercritical conditions with CO₂ containing a high amount ofa polar solvent such as ethanol to extract a polar lipid fractioncontaining ether and non-ether phospholipids; and then blending theneutral and polar lipid extracts to provide the compositions describedabove.

In some embodiments, the compositions of this invention are contained inacceptable excipients and/or carriers for oral consumption. The actualform of the carrier, and thus, the composition itself, is not critical.The carrier may be a liquid, gel, gelcap, capsule, powder, solid tablet(coated or non-coated), tea, or the like. The composition is preferablyin the form of a tablet or capsule and most preferably in the form of asoft gel capsule. Suitable excipient and/or carriers includemaltodextrin, calcium carbonate, dicalcium phosphate, tricalciumphosphate, microcrystalline cellulose, dextrose, rice flour, magnesiumstearate, stearic acid, croscarmellose sodium, sodium starch glycolate,crospovidone, sucrose, vegetable gums, lactose, methylcellulose,povidone, carboxymethylcellulose, corn starch, and the like (includingmixtures thereof). Preferred carriers include calcium carbonate,magnesium stearate, maltodextrin, and mixtures thereof. The variousingredients and the excipient and/or carrier are mixed and formed intothe desired form using conventional techniques. The tablet or capsule ofthe present invention may be coated with an enteric coating thatdissolves at a pH of about 6.0 to 7.0. A suitable enteric coating thatdissolves in the small intestine but not in the stomach is celluloseacetate phthalate. Further details on techniques for formulation for andadministration may be found in the latest edition of Remington'sPharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).

The composition may comprise one or more inert ingredients, especiallyif it is desirable to limit the number of calories added to the diet bythe composition. For example, the dietary supplement of the presentinvention may also contain optional ingredients including, for example,herbs, vitamins, minerals, enhancers, colorants, sweeteners, flavorants,inert ingredients, and the like. For example, the composition of thepresent invention may contain one or more of the following: ascorbates(ascorbic acid, mineral ascorbate salts, rose hips, acerola, and thelike), dehydroepiandosterone (DHEA), Fo-Ti or Ho Shu Wu (herb common totraditional Asian treatments), Cat's Claw (ancient herbal ingredient),green tea (polyphenols), inositol, kelp, dulse, bioflavinoids,maltodextrin, nettles, niacin, niacinamide, rosemary, selenium, silica(silicon dioxide, silica gel, horsetail, shavegrass, and the like),spirulina, zinc, and the like. Such optional ingredients may be eithernaturally occurring or concentrated forms.

In some embodiments, the composition may further comprise vitamins andminerals including, but not limited to, calcium phosphate or acetate,tribasic; potassium phosphate, dibasic; magnesium sulfate or oxide; salt(sodium chloride); potassium chloride or acetate; ascorbic acid; ferricorthophosphate; niacinamide; zinc sulfate or oxide; calciumpantothenate; copper gluconate; riboflavin; beta-carotene; pyridoxine;folic acid; thiamine; biotin; chromium chloride or picolonate; potassiumiodide; sodium selenate; sodium molybdate; phylloquinone; retinoic acid;cyanocobalamin; sodium selenite; copper sulfate; vitamin A; vitamin C;inositol; potassium iodide; vitamin E, vitamin K; niacin; andpantothenic acid. Suitable dosages for vitamins and minerals may beobtained, for example, by consulting the U.S. RDA guidelines. In stillother embodiments, the particles comprise an amino acid supplementformula in which at least one amino acid is included (e.g., 1-carnitineor tryptophan).

In further embodiments, the composition comprises at least one foodflavoring such as acetaldehyde (ethanal), acetoin (acetylmethylcarbinol), anethole (parapropenyl anisole), benzaldehyde (benzoicaldehyde), N butyric acid (butanoic acid), d or I carvone (carvol),cinnamaldehyde (cinnamic aldehyde), citral (3,7-dimethyl-2,6-octadienal,geranial, neral), decanal (N-decylaldehyde, capraldehyde, capricaldehyde, caprinaldehyde, aldehyde C 10), ethyl acetate, ethyl butyrate,3-methyl-3-phenyl glycidic acid ethyl ester (ethyl methyl phenylglycidate, strawberry aldehyde, C₁₋₆ aldehyde), ethyl vanillin, geraniol(3,7-dimethylocta-2,6-dien-1-ol), geranyl acetate (geraniol acetate),limonene (d, l, and dl), linalool (3,7-dimethylocta-1,6-dien-3-ol),linalyl acetate (bergamot), methyl anthranilate(methyl-2-aminobenzoate), piperonal (3,4-methylenedioxy benzaldehyde,heliotropin), vanillin, alfalfa (Medicago sativa L.), allspice (Pimentaofficinalis), ambrette seed (Hibiscus abelmoschus), angelic (Angelicaarchangelica), Angostura (Galipea officinalis), anise (Pimpinefiaanisum), star anise (Illicium verum), balm (Melissa officinalis), basil(Ocimum basilicum), bay (Laurus nobilis), calendula (Calendulaofficinalis), chamomile (Anthemis nobilis), capsicum (Capsicumfrutescens), caraway (Carum carvi), cardamom (Elettaria cardamomum),cassia (Cinnamomum cassia), cayenne pepper (Capsicum frutescens), Celeryseed (Apium graveolens), chervil (Anthriscus cerefolium), chives (Alliumschoenoprasum), coriander (Coriandrum sativum), cumin (Cuminum cyminum),elder flowers (Sambucus canadensis), fennel (Foeniculum vulgare),fenugreek (Trigonella foenum graecum), ginger (Zingiber officinale),horehound (Marrubium vulgare), horseradish (Armoracia lapathifolia),hyssop (Hyssopus officinalis), lavender (Lavandula officinalis), mace(Myristica fragrans), marjoram (Majorana hortensis), mustard (Brassicanigra, Brassica juncea, Brassica hirta), nutmeg (Myristica fragrans),paprika (Capsicum annuum), black pepper (Piper nigrum), peppermint(Mentha piperita), poppy seed (Papayer somniferum), rosemary (Rosmarinusofficinalis), saffron (Crocus sativus), sage (Salvia officinalis),savory (Satureia hortensis, Satureia montana), sesame (Sesamum indicum),spearmint (Mentha spicata), tarragon (Artemisia dracunculus), thyme(Thymus vulgaris, Thymus serpyllum), turmeric (Curcuma longa), vanilla(Vanilla planifolia), zedoary (Curcuma zedoaria), sucrose, glucose,saccharin, sorbitol, mannitol, aspartame. Other suitable flavoring aredisclosed in such references as Remington's Pharmaceutical Sciences,18th Edition, Mack Publishing, p. 1288-1300 (1990), and Furia andPellanca, Fenaroli's Handbook of Flavor Ingredients, The Chemical RubberCompany, Cleveland, Ohio, (1971), known to those skilled in the art.

In other embodiments, the compositions comprise at least one syntheticor natural food coloring (e.g., annatto extract, astaxanthin, beetpowder, ultramarine blue, canthaxanthin, caramel, carotenal, betacarotene, carmine, toasted cottonseed flour, ferrous gluconate, ferrouslactate, grape color extract, grape skin extract, iron oxide, fruitjuice, vegetable juice, dried algae meal, tagetes meal, carrot oil, cornendosperm oil, paprika, paprika oleoresin, riboflavin, saffron, tumeric,and oleoresin).

In still further embodiments, the compositions comprise at least onephytonutrient (e.g., soy isoflavonoids, oligomeric proanthcyanidins,indol-3-carbinol, sulforaphone, fibrous ligands, plant phytosterols,ferulic acid, anthocyanocides, triterpenes, omega 3/6 fatty acids,conjugated fatty acids such as conjugated linoleic acid and conjugatedlinolenic acid, polyacetylene, quinones, terpenes, cathechins, gallates,and quercitin). Sources of plant phytonutrients include, but are notlimited to, soy lecithin, soy isoflavones, brown rice germ, royal jelly,bee propolis, acerola berry juice powder, Japanese green tea, grape seedextract, grape skin extract, carrot juice, bilberry, flaxseed meal, beepollen, ginkgo biloba, primrose (evening primrose oil), red clover,burdock root, dandelion, parsley, rose hips, milk thistle, ginger,Siberian ginseng, rosemary, curcumin, garlic, lycopene, grapefruit seedextract, spinach, and broccoli.

EXAMPLES

The present invention is further described in the following non-limitingExamples.

Example 1

Antarctic krill (Euphausia superba) was captured and brought on boardalive, before it was processed into krill meal, an oil (asta oil), andstickwater. During the krill meal processing a neutral oil (asta oil) isrecovered.

Example 2

The krill meal obtained in Example 1 was then ethanol extractedaccording to the method disclosed in JP 02-215351, the contents of whichare incorporated herein by reference. The results showed that around 22%fat from the meal could be extracted. Table 1 shows the fatty acidcomposition of the krill meal and the krill oil extracted from the mealusing ethanol. Table 2 shows the composition and properties of the krillmeal and products before and after extraction, whereas Table 3 shows thelipid composition.

TABLE 1 Fatty acid distribution in krill meal (g/100 g lipid) and theethanol extracted krill oil. Fatty Acid File Krill meal EtOH KO C4:00.00 C6:0 0.00 C8:0 0.00 C10:0 0.00 C12:0 0.00 C14:0 7.8 6.4 C14:1 0.00C15:0 0.00 C16:0 15.8 14.7 C16:1 5.1 4.2 C18:0 0.9 0.7 C18:1 13.4 11.8C18:2N6 1.1 1.2 C18:3N6 0.1 0.1 C18:3N3 0.4 0.4 C18:4N3 1.1 0.1 C20:00.1 0.1 C20:1 0.8 0.6 C20:2N6 <0.1 <0.1 C20:3N6 0.1 <0.1 C20:4N6 0.2 0.2C20:3N3 <0.1 <0.1 C20:4N3 0.2 0.2 C20:5N3 (EPA) 10.5 10.4 C22:0 <0.1<0.1 C22:1 0.5 0.4 C22:2N6 <0.1 <0.1 C22:4N6 <0.1 C22:5N6 0.00 C22:5N30.2 C22:6N3 (DHA) 5.4 4.8 C24:1 0.03 Saturated 24.6 21.9 Monounsaturated19.9 17.0 Polyunsaturated 21.0 19.4 Total 65.5 58.2 Omega-3 18.2 17.0Omega-6 1.3

TABLE 2 Composition and properties of the krill meal and products afterextraction Delipidated EtOH extracted Krill Meal krill meal krill oilCrude protein 586 g/kg 735 g/kg Fat (Folch) 250 g/kg 30 g/kgMoisture/ethanol 71 g/kg 134 g/kg 85 g/kg Astaxanthin esters 144 mg/kg10 mg/kg 117 mg/kg Diesters 110 mg/kg 8.5 mg/kg 117 mg/kg Monoesters 33mg/kg 1.8 mg/kg 37 mg/kg Biological 854 g/kg 870 g/kg digestible proteinprotein Flow number 4.8 1.9 NH3 9 mg N/100 g 0 3 mg N/100 g TMA 2 mgN/100 g 0 70 mg N/100 g TMAO 125 mg N/100 g 0 456 mg N/100 g

TABLE 3 Lipid class distribution Delipidated EtOH Krill meal krill mealextracted KO Cholesterol ester 3.5 TG 32.7 37.4 31.1 FFA 7.8 14.1 16.0Cholesterol 9.1 8.0 12.6 DG 1.1 3.3 MG 3.7 Sphingolipid 2.8 PE 6.5 2.52.7 Cardiolipin 4.2 PI 1.1 11.0 PS 1.4 PC 28.6 20.2 25.3 LPC 2.9 2.6 6.2Total polar lipids 40.6 40.5 36.9 Total neutral lipids 54.2 59.5 63.1

Example 3

The krill meal obtained in Example 1 was then subjected to asupercritical fluid extraction method in two stages. During stage 1,12.1% fat (neutral krill oil) was removed using neat CO₂ only at 300bars, 60° C. and for 30 minutes. In stage 2, the pressure was increasedto 400 bar and 20% ethanol was added (v/v) for 90 minutes. This resultedin further extraction of 9% polar fat which hereafter is called polarkrill oil. The total fatty acid composition of the polar krill oil, theneutral krill oil and a commercial product obtained from Neptune Biotech(Laval, Quebec, Canada) are listed in Table 4. In addition the fattyacid composition for the phospholipids (Table 5), the neutral lipids(Table 6), the free fatty acids, diglycerides (Table 7), triglycerides,lyso-phosphatidylcholine (LPC) (Table 8), phosphatidylcholine (PC),phosphatidylethanolamine (PE) (Table 9), phosphatidylinositol (PI) andphosphatidylserine (PS) (Table 10) are shown. Table 11 shows the levelof astaxanthin and cholesterol for the different fractions.

TABLE 4 Total fatty acids compostions of the krill oil products (%(w/w)) Total Fatly Acids Fatty Acid Neutral Polar File KO KO NKO C4:00.00 0.00 0.00 C6:0 0.00 0.00 0.00 C8:0 0.00 0.00 0.00 C10:0 0.00 0.000.00 C12:0 0.47 0.04 0.24 C14:0 22.08 3.28 12.48 C14:1 0.33 0.01 0.17C15:0 0.58 0.36 0.52 C16:0 27.03 29.25 23.25 C16:1 0.07 0.01 8.44 C18:01.72 1.03 1.42 C18:1 30.29 13.57 18.92 C18:2N6 2.10 1.96 1.71 C18:3N60.30 0.21 0.00 C18:3N3 0.69 1.02 1.32 C18:4N3 0.05 1.81 3.50 C20:0 0.060.00 0.05 C20:1 1.87 0.80 1.16 C20:2N6 0.05 0.05 0.05 C20:3N6 0.22 0.730.04 C20:4N6 0.00 0.00 0.49 C20:3N3 0.09 0.09 0.06 C20:4N3 0.24 0.510.33 C20:5N3 (EPA) 7.33 29.88 16.27 C22:0 0.01 0.06 0.05 C22:1 0.64 1.780.82 C22:2N6 0.00 0.00 0.00 C22:4N6 0.00 0.00 0.07 C22:5N6 0.00 0.030.00 C22:5N3 0.21 0.67 0.36 C22:6N3 (DHA) 3.51 12.61 8.17 C24:0 0.050.00 0.01 C24:1 0.03 0.25 0.11 Total 100.00 100.00 100.00 Saturated52.00 34.01 38.01 Monounsaturated 33.22 16.43 29.61 Polyunsaturated14.77 49.56 32.37 Total 100.00 100.00 100.00 Omega-3 12.11 46.58 30.02Omega-6 2.67 2.98 2.35

TABLE 5 Fatty acid composition of the phospholipid fraction (% (w/w)).Total Phospholipid Fatty Acid Neutral Polar Neptune File KO KO KO C4:00.00 0.00 0.00 C6:0 0.00 0.00 0.00 C8:0 0.00 0.00 0.00 C10:0 0.00 0.000.00 C12:0 0.00 0.00 0.00 C14:0 0.01 0.00 0.00 C14:1 0.42 0.01 0.01C15:0 2.52 0.00 0.00 C16:0 4.73 35.78 32.81 C16:1 0.19 0.17 0.19 C18:06.31 1.18 1.55 C18:1 38.40 15.58 13.54 C18:2N6 4.18 2.16 1.90 C18:3N60.18 0.22 0.19 C18:3N3 1.02 1.05 1.48 C18:4N3 3.08 1.62 2.15 C20:0 0.270.00 0.07 C20:1 2.55 1.02 0.78 C20:2N6 0.19 0.06 0.06 C20:3N6 0.00 0.140.10 C20:4N6 0.57 0.62 0.64 C20:3N3 0.43 0.08 0.09 C20:4N3 0.17 0.450.42 C20:5N3 (EPA) 20.58 25.53 26.47 C22:0 0.14 0.06 0.00 C22:1 0.002.09 1.94 C22:2N6 0.25 0.71 0.85 C22:4N6 0.44 0.00 0.03 C22:5N6 0.110.00 0.00 C22:5N3 0.00 0.60 0.63 C22:6N3 (DHA) 10.93 10.30 13.34 C24:01.77 0.30 0.37 C24:1 0.59 0.28 0.38 Total 100.00 100.00 100.00 Saturated15.74 37.32 34.81 Monounsaturated 42.14 19.15 16.84 Polyunsaturated42.12 43.53 48.34 Total 100.00 100.00 100.00 Omega-3 36.22 39.62 44.56Omega-6 5.91 3.90 3.78

TABLE 6 Fatty acid composition of the total neutral lipid fraction (%(w/w)). Total neutral lipid Fatty Acid Neutral Polar Neptune File KO KOKO C4:0 0.00 0.00 0.00 C6:0 0.00 0.00 0.00 C8:0 0.00 0.00 0.00 C10:00.00 0.00 0.00 C12:0 0.00 0.00 0.00 C14:0 20.35 11.31 18.44 C14:1 0.300.29 0.25 C15:0 0.53 1.53 0.62 C16:0 23.79 0.49 24.11 C16:1 12.42 5.2211.86 C18:0 1.54 3.27 1.67 C18:1 26.81 33.09 23.82 C18:2N6 1.68 2.371.79 C18:3N6 0.20 0.23 0.25 C18:3N3 0.59 0.62 0.03 C18:4N3 0.03 1.270.05 C20:0 0.07 0.00 0.06 C20:1 1.63 1.41 1.39 C20:2N6 0.04 0.00 0.05C20:3N6 0.18 0.94 0.01 C20:4N6 0.00 0.00 0.00 C20:3N3 0.09 0.00 0.01C20:4N3 0.18 0.41 0.23 C20:5N3 (EPA) 5.88 19.26 9.68 C22:0 0.02 0.000.03 C22:1 0.56 0.60 0.53 C22:2N6 0.00 0.00 0.00 C22:4N6 0.00 0.00 0.04C22:5N6 0.01 0.00 0.00 C22:5N3 0.17 0.27 0.22 C22:6N3 (DHA) 2.74 17.224.64 C24:0 0.15 0.00 0.17 C24:1 0.03 0.21 0.09 Total 100.00 100.00100.00 Saturated 46.45 16.60 45.10 Monounsaturated 41.75 40.82 37.91Polyunsaturated 11.80 42.59 16.99 Total 100.00 100.00 100.00 Omega-39.68 39.05 14.86 Omega-6 2.11 3.54 2.14

TABLE 7 Fatty acid composition of the diglyceride and free fatty acids(% (w/w)). Diglycerides Free fatty acids Fatty Neutral Polar NeptuneNeutral Polar Neptune Acid File KO KO KO KO KO KO C4:0 0.00 0.00 0.000.00 0.00 0.00 C6:0 0.00 0.00 0.00 0.00 0.00 0.00 C8:0 0.00 0.00 0.000.00 0.00 0.00 C10:0 0.00 0.00 0.00 0.00 0.00 0.00 C12:0 0.00 0.00 0.000.00 0.00 0.00 C14:0 13.85 14.35 12.22 5.86 7.19 5.45 C14:1 0.18 0.000.17 0.05 0.00 0.08 C15:0 0.49 1.08 0.66 0.46 1.60 0.45 C16:0 23.6835.24 25.81 28.30 29.37 21.12 C16:1 9.49 6.80 0.09 3.27 3.08 4.91 C18:01.56 3.63 1.89 1.13 2.43 0.99 C18:1 23.67 19.85 23.82 14.50 14.77 17.41C18:2N6 1.79 0.21 1.90 1.69 0.97 1.86 C18:3N6 0.17 0.00 0.01 0.14 0.000.22 C18:3N3 0.69 0.00 1.19 0.85 0.00 1.34 C18:4N3 1.92 0.00 2.75 1.300.00 2.72 C20:0 0.00 0.00 0.00 0.00 0.00 0.00 C20:1 1.09 0.00 1.01 0.480.00 0.57 C20:2N6 0.00 0.00 0.00 0.00 0.00 0.00 C20:3N6 0.13 0.00 0.000.08 0.00 0.05 C20:4N6 0.45 0.00 0.64 0.78 0.00 1.43 C20:3N3 0.00 0.000.00 0.00 0.00 0.00 C20:4N3 0.35 0.00 0.43 0.39 0.00 0.43 C20:5N3 14.039.80 18.00 24.33 23.57 25.36 (EPA) C22:0 0.18 0.00 0.10 0.00 0.00 0.05C22:1 0.41 0.00 0.57 0.80 0.69 0.37 C22:2N6 0.28 0.00 0.50 0.46 0.000.54 C22:4N6 0.00 0.00 0.00 0.00 0.00 0.00 C22:5N6 0.00 0.00 0.00 0.000.00 0.00 C22:5N3 0.20 0.00 0.27 0.34 0.00 0.32 C22:6N3 4.74 9.04 7.5314.31 16.33 13.95 (DHA) C24:0 0.64 0.00 0.42 0.49 0.00 0.39 C24:1 0.000.00 0.00 0.00 0.00 0.00 Total 100.00 100.00 100.00 100.00 100.00 100.00Saturated 40.40 54.30 41.10 36.24 40.59 28.45 Monoun- 34.84 26.64 25.6619.09 18.54 23.34 saturated Polyun- 24.77 19.06 33.24 44.67 40.87 48.22saturated Total 100.00 100.00 100.00 100.00 100.00 100.00 Omega-3 21.9518.85 30.18 41.51 39.90 44.13 Omega-6 2.82 0.21 3.05 3.15 0.97 4.09

TABLE 8 Fatty acid composition of the triglyceride andlyso-phophatidylcholine fractions (% (w/w)). Triglycerides Lyso PC FattyNeutral Polar Neptune Neutral Polar Neptune Acid File KO KO KO KO KO KOC4:0 0.00 0.00 0.00 0.00 0.00 0.00 C6:0 0.00 0.00 0.00 0.00 0.00 0.00C8:0 0.00 0.00 0.00 0.00 0.00 0.00 C10:0 0.00 0.00 0.00 0.00 0.00 0.00C12:0 0.00 0.00 0.00 0.00 0.00 0.00 C14:0 23.06 26.65 25.13 19.38 4.272.87 C14:1 0.36 0.93 0.36 0.00 0.08 0.00 C15:0 0.56 2.64 0.78 0.00 0.520.45 C16:0 23.17 4.93 27.80 41.00 44.14 30.56 C16:1 13.68 11.58 0.040.00 1.84 2.24 C18:0 1.52 3.12 1.99 0.76 1.59 1.32 C18:1 27.83 34.3927.92 6.65 14.24 11.29 C18:2N6 1.64 2.05 1.92 0.00 1.75 2.07 C18:3N60.20 0.00 0.30 0.00 0.00 0.06 C18:3N3 0.51 0.00 0.00 7.95 0.67 1.75C18:4N3 1.99 0.00 4.83 0.00 1.11 2.46 C20:0 0.06 0.00 0.08 0.00 0.000.00 C20:1 1.67 0.00 1.76 0.00 0.52 0.00 C20:2N6 0.04 0.00 0.05 0.000.00 0.00 C20:3N6 0.05 0.00 0.01 0.00 0.00 0.54 C20:4N6 0.00 0.00 0.000.00 0.40 0.00 C20:3N3 0.05 0.00 0.07 0.00 0.00 0.00 C20:4N3 0.11 0.000.17 0.00 0.31 0.55 C20:5N3 2.10 7.97 4.44 0.00 18.59 28.48 (EPA) C22:00.02 0.00 0.04 0.00 0.00 0.00 C22:1 0.37 0.00 0.42 0.00 1.46 0.91C22:2N6 0.00 0.00 0.00 0.00 0.00 0.00 C22:4N6 0.01 0.00 0.01 0.00 0.000.00 C22:5N6 0.00 0.00 0.01 0.00 0.00 0.00 C22:5N3 0.10 0.00 0.16 0.000.41 0.62 C22:6N3 0.67 3.97 1.42 24.26 7.79 13.82 (DHA) C24:0 0.26 1.780.26 0.00 0.32 0.00 C24:1 0.00 0.00 0.03 0.00 0.00 0.00 Total 100.00100.00 100.00 100.00 100.00 100.00 Saturated 48.64 39.12 56.08 61.1450.83 35.21 Monoun- 43.90 46.89 30.52 6.65 18.14 14.44 saturated Polyun-7.45 13.99 13.41 32.20 31.02 50.35 saturated Total 100.00 100.00 100.00100.00 100.00 100.00 Omega-3 5.51 11.94 11.11 32.20 28.87 47.69 Omega-61.94 2.05 2.30 0.00 2.15 2.66

TABLE 9 Fatty acid composition of the phosphatidylcholine and thephosphatidylserine fractions (% (w/w)). PC PS Fatty Neutral PolarNeptune Neutral Polar Neptune Acid File KO KO KO KO KO KO C4:0 0.00 0.000.00 0.00 0.00 0.00 C6:0 0.00 0.00 0.00 0.00 0.00 0.00 C8:0 0.00 0.000.00 0.00 0.00 0.00 C10:0 0.00 0.00 0.00 0.00 0.00 0.00 C12:0 0.00 0.000.00 0.00 0.00 0.00 C14:0 0.75 3.29 2.77 7.60 9.52 2.31 C14:1 2.07 0.040.02 0.00 0.00 0.00 C15:0 1.34 0.00 0.00 3.83 0.00 0.00 C16:0 16.6531.92 29.83 30.44 43.61 19.49 C16:1 0.96 0.01 0.17 9.96 3.47 2.79 C18:01.33 1.06 1.33 2.08 3.34 2.24 C18:1 34.34 13.55 11.16 0.00 7.37 11.87C18:2N6 10.55 2.27 1.90 0.00 0.00 0.00 C18:3N6 1.44 0.25 0.20 0.00 0.000.00 C18:3N3 2.49 1.19 1.54 0.00 0.00 0.00 C18:4N3 2.38 1.92 2.41 0.000.00 0.00 C20:0 2.79 0.03 0.05 0.00 0.00 0.00 C20:1 2.42 0.82 0.74 0.000.00 0.00 C20:2N6 0.56 0.05 0.06 0.00 0.00 0.00 C20:3N6 0.67 0.13 0.090.00 0.00 0.00 C20:4N6 1.85 0.61 0.56 0.00 0.00 0.00 C20:3N3 3.94 0.070.06 0.00 0.00 0.33 C20:4N3 4.32 0.50 0.46 0.00 0.00 0.00 C20:5N3 1.0829.85 30.09 25.84 15.81 16.35 (EPA) C22:0 0.00 0.05 0.02 0.00 0.00 0.00C22:1 2.77 0.00 1.87 0.00 0.00 0.00 C22:2N6 0.00 0.81 0.97 0.00 0.000.00 C22:4N6 0.00 0.01 0.02 0.00 0.00 0.00 C22:5N6 1.49 0.01 0.00 0.000.00 0.00 C22:5N3 1.48 0.67 0.68 0.00 0.00 0.00 C22:6N3 0.00 10.53 12.4920.25 16.89 44.63 (DHA) C24:0 2.34 0.10 0.18 0.00 0.00 0.00 C24:1 0.000.25 0.34 0.00 0.00 0.00 Total 100.00 100.00 100.00 100.00 100.00 100.00Saturated 25.19 36.46 34.18 43.95 56.47 24.04 Monoun- 42.56 14.67 14.299.96 10.84 14.65 saturated Polyun- 32.25 48.87 51.53 46.09 32.69 61.31saturated Total 100.00 100.00 100.00 100.00 100.00 100.00 Omega-3 15.6944.73 47.73 46.09 32.69 61.31 Omega-6 16.56 4.13 3.81 0.00 0.00 0.00C4:0 0.00 0.00 0.00 0.00 0.00 0.00 C6:0 0.00 0.00 0.00 0.00 0.00 0.00C8:0 0.00 0.00 0.00 0.00 0.00 0.00 C10:0 0.00 0.00 0.00 0.00 0.00 0.00C12:0 0.00 0.00 0.00 0.00 0.00 0.00 C14:0 11.15 5.82 5.72 14.42 4.600.83 C14:1 3.03 0.66 0.00 0.00 0.00 0.10 C15:0 5.86 1.95 3.18 0.00 1.300.23 C16:0 37.02 30.66 31.39 35.91 31.21 18.38 C16:1 18.05 2.24 1.160.00 1.51 0.75 C18:0 6.72 2.83 5.56 12.72 16.70 1.84 C18:1 18.15 24.7714.23 36.96 19.91 18.45

TABLE 10 Fatty acid composition of the phosphatidylinositol andphophatidylethanolamine fractions (% (w/w)). PI PE Fatty Neutral PolarNeptune Neutral Polar Neptune Acid File KO KO KO KO KO KO C18:2N6 0.002.67 0.00 0.00 2.62 0.85 C18:3N6 0.00 0.00 0.00 0.00 0.00 0.00 C18:3N30.00 0.00 0.00 0.00 0.00 0.33 C18:4N3 0.00 0.00 0.00 0.00 0.00 0.00C20:0 0.00 0.00 0.00 0.00 0.00 0.00 C20:1 0.00 0.00 0.00 0.00 0.00 0.00C20:2N6 0.00 0.00 0.00 0.00 0.00 0.00 C20:3N6 0.00 0.00 0.00 0.00 0.001.15 C20:4N6 0.00 0.00 0.00 0.00 0.00 0.00 C20:3N3 0.00 0.00 0.00 0.000.00 0.00 C20:4N3 0.00 0.00 0.00 0.00 0.00 0.00 C20:5N3 0.00 17.60 20.450.00 10.76 21.26 (EPA) C22:0 0.00 0.00 0.00 0.00 0.00 0.00 C22:1 0.000.00 0.00 0.00 0.00 0.00 C22:2N6 0.00 0.00 0.00 0.00 0.00 0.00 C22:4N60.00 0.00 0.00 0.00 0.00 0.00 C22:5N6 0.00 0.00 0.00 0.00 0.00 0.00C22:5N3 0.00 0.00 0.00 0.00 0.00 0.67 C22:6N3 0.00 10.79 18.32 0.0011.39 35.16 (DHA) C24:0 0.00 0.00 0.00 0.00 0.00 0.00 C24:1 0.00 0.000.00 0.00 0.00 0.00 Total 100.00 100.00 100.00 100.00 100.00 100.00Saturated 60.76 41.26 45.84 63.04 53.81 21.28 Monoun- 39.24 27.67 15.3936.96 21.42 19.30 saturated Polyun- 0.00 31.07 38.77 0.00 24.77 59.42saturated Total 100.00 100.00 100.00 100.00 100.00 100.00 Omega-3 0.0028.40 38.77 0.00 22.15 57.43 Omega-6 0.00 2.67 0.00 0.00 2.62 1.99

TABLE 11 Compositional data for the novel krill oil composition obtainedand NKO krill oil. Ethanol Neptune extracted Polar Neutral Compounds KOKO KO KO Astaxanthin 472 mg/kg 117 mg/kg 580 mg/kg 98 mg/kg estersAstaxanthin 11 mg/kg <1 mg/kg <1 mg/kg <1 mg/kg free Total 1 g/100 g 12g/100 g <0.5 g/100 g 5.7 g/100 g cholesterol

Example 4

Neutral lipids were extracted from krill meal (138 kg) using SFE withneat CO₂ (solvent ratio 25 kg/kg) at 500 bar and 75° C. The neutrallipids were fractionated at 200 bar (75° C.) and at 60 bar (35° C.) attwo separators. The extract obtained at the first separator (S1-19.6 kg)was characterized, and the results can be found in Table 12. The extractobtained at the second separator (S2-0.4 kg) was rich in water, and wasnot further used. Next, the polar lipids were extracted using CO₂ at 500bar, 20% ethanol and at a temperature of 75° C. Using a solvent ratio of32 (kg/kg) and collecting an extract of 18.2 kg using a separator at 60bars and 35° C. The polar lipids were collected and analyzed (see Table13). Next, the polar lipids were mixed in a 50/50 ratio with the neutrallipids collected from the first separator before finally the ethanol wasremoved carefully by evaporation. The product obtained was red andtransparent. If the ethanol is removed before the mixing if thefractions a transparent product is not obtained. The composition of the50/50 red and transparent product can be found in Table 14.

TABLE 12 Fatty acid composition of the extract collected in S1 Fattyacid Unit Amount 14:0 g/100 g 18.4 16:0 g/100 g 22.2 18:0 g/100 g 1.516:1 n-7 g/100 g 10.9 18:1 (n-9) + (n-7) + (n-5) g/100 g 25.6 20:1(n-9) + (n-7) g/100 g 1.8 22-1 (n-11) + (n-9) + (n-7) g/100 g 0.5 16:2(n-4) g/100 g 1.3 16:4 (n-1) g/100 g 1.2 18:2 n-6 g/100 g 1.3 18:3 n-3g/100 g 0.8 18:4 n-3 g/100 g 2.9 20:5 n-3 g/100 g 4.1 22:6 n-4 g/100 g1.7 Lipid class composition of the extract collected in S1 Lipid UnitAmount Triacylglycerol g/100 g 84 Diacylglycerol g/100 g 0.7 Free fattyacids g/100 g 1.3 Cholesterol g/100 g 2.7 Cholesterol esters g/100 g 0.9Miscellaneous analysis of the extract in S1. Compound Unit Amount Freeastaxanthin mg/kg 4.3 Astaxanthin esters mg/kg 462 Trimethylamin mgN/100 g <1 Trimethylamineoxide mg N/100 g 2

TABLE 13 Fatty acid composition of the extract collected after CO₂ and20% etanol in S1. Fatty acid Unit Amount 14:0 g/100 g 1.3 16:0 g/100 g13.8 18:0 g/100 g 0.6 16:1 n-7 g/100 g 0.9 18:1 (n-9) + (n-7) + (n-5)g/100 g 6.5 20:1 (n-9) + (n-7) g/100 g 0.6 22:1 (n-11) + (n-9) + (n-7)g/100 g 0.1 16:2 (n-4) g/100 g <0.1 16:4 (n-1) g/100 g <0.1 18:2 n-6g/100 g 0.8 18:3 n-3 g/100 g 0.6 18:4 n-3 g/100 g 1.0 20:5 n-3 g/100 g14.7 22:6 n-4 g/100 g 6.5 Lipid class composition of the extractcollected after CO₂ and 20% etanol in S1. Lipid Unit AmountTriacylglycerol g/100 g <0.5 Cholesterol g/100 g <0.5Phophatidylethanolamine g/100 g 1.6 Phosphatidylcholine g/100 g 67Lyso-phophatidylcholine g/100 g 4.4 Miscellaneous analysis of theextract in S1. Compound Unit Amount Trimethylamin mg N/100 g 422Trimethylamineoxide mg N/100 g 239

TABLE 14 Fatty acid composition of the final blended product obtained inExample 4 in S1. Fatty acid Unit Amount 14:0 g/100 g 9.7 16:0 g/100 g18.5 18:0 g/100 g 1.0 16:1 n-7 g/100 g 5.8 18:1 (n-9) + (n-7) + (n-5)g/100 g 16.0 20:1 (n-9) + (n-7) g/100 g 1.2 22:1 (n-11) + (n-9) + (n-7)g/100 g 1.0 16:2 (n-4) g/100 g 0.3 16:4 (n-1) g/100 g <0.1 18:2 n-6g/100 g 1.0 18:3 n-3 g/100 g 0.8 18:4 n-3 g/100 g 2.1 20:5 n-3 g/100 g10.7 22:6 n-4 g/100 g 4.7 Lipid class composition of the final blendedproduct obtained in Example 4. Lipid Unit Amount Triacylglycerol g/100 g53 Diacylglycerol g/100 g 1.3 Free fatty acids g/100 g 0.5 Cholesterolg/100 g 0.6 Cholesterol esters g/100 g <0.5 Phophatidylethanolamineg/100 g <1 Phosphatidylcholine g/100 g 42 Lyso-phophatidylcholine g/100g 5.9 Miscellaneous analysis of the final blended product obtained inexample 4. Compound Unit Amount Free astaxanthin mg/kg 1.1 Astaxanthinesters mg/kg 151 Trimethylamin mg N/100 g 109 Trimethylamineoxide mgN/100 g 80

Example 5

The asta oil obtained in Example 1 was blended with the polar lipidsobtained in Example 4 in a ratio of 46:54 (v/v). Next the ethanol wasremoved by evaporation and a dark red and transparent product wasobtained. The product was analyzed and the results can be found in Table15. Furthermore, the product was encapsulated into soft gelssuccessfully. During the encapsulation it was observed that any furtherincrease in phospholipids, and thereby viscosity, will make it verydifficult to encapsulate the final product.

TABLE 15 Fatty acid composition of the final blended product obtained inExample 5. Fatty acid Unit Amount 14:0 g/100 g 8.2 16:0 g/100 g 17.718:0 g/100 g 1.0 16:1 n-7 g/100 g 4.9 18:1 (n-9) + (n-7) + (n-5) g/100 g14.9 20:1 (n-9) + (n-7) g/100 g 1.1 22:1 (n-11) + (n-9) + (n-7) g/100 g1.0 16:2 (n-4) g/100 g 0.4 16:4 (n-1) g/100 g <0.1 18:2 n-6 g/100 g 1.218:3 n-3 g/100 g 0.8 18:4 n-3 g/100 g 1.8 20:5 n-3 g/100 g 10.6 22:6 n-4g/100 g 4.8 Lipid class composition of the final blended productobtained in Example 5. Lipid Unit Amount Triacylglycerol g/100 g 41Diacylglycerol g/100 g 0.8 Free fatty acids g/100 g 1.2 Cholesterolg/100 g 0.4 Cholesterol esters g/100 g 0.3 Phophatidylethanolamine g/100g 0.6 Phosphatidylcholine g/100 g 51 Lyso-phophatidylcholine g/100 g<0.5 Total polar lipids g/100 g 52.4 Total neutral lipids g/100 g 43.6Miscellaneous analysis of the final blended product obtained in Example5 Compound Unit Amount Free astaxanthin mg/kg 12 Astaxanthin estersmg/kg 1302 Trimethylamin mg N/100 g 193 Trimethylamineoxide mg N/100 g1.7

Example 6

Krill lipids were extracted from krill meal (a food grade powder) usingsupercritical fluid extraction with co-solvent. Initially, 300 barpressure, 333° K and 5% ethanol (ethanol: CO₂, w/w) were utilized for 60minutes in order to remove neutral lipids and astaxanthin from the krillmeal. Next, the ethanol content was increased to 23% and the extractionwas maintained for 3 hours and 40 minutes. The extract was thenevaporated using a falling film evaporator and the resulting krill oilwas finally filtered. The product obtained was then analyzed and theresults can be found in Table 16.

TABLE 16 Analysis of the krill oil obtained using supercritical fluidextraction. Parameter Value Ethanol 1.11% w/w Water Content 2.98% w/wC20:5 n-3 (EPA) 19.9 C22:6 n-3 (DHA) 11.3 Total Omega 3 35.7 Total Omega6  3.0 Total Phospholipids 50.55 wt % Ratio Omega3-PL/Total Omega 377.6% w/w Ratio EPA-PL/Total EPA 84.4% w/w Ratio DHA-PL/Total DHA 74.7%w/w Triglycerides 25.9 g/100 g Astaxanthin 2091 mg/kg Peroxide Value<0.1

Example 7

Krill oil was prepared according to the method described in Example 6 byextracting from the same krill meal. The oil was subjected to ³¹P NMRanalysis for the identification and quantification of the various formsof phospholipids. The analysis was performed according to the followingmethods: Samples (20-40 mg) were weighed into 1.5 ml centrifuge tubes.Next, NMR detergent (750 μl-10% Na cholate, 1% EDTA, pH 7.0 in H₂O+D₂O,0.3 g L-1 PMG internal standard) was added. Next, the tube was placed inan oven at 60° C. and periodically shaken/sonicated until completelydispersed. The solution was then transferred to a 5 ml NMR tube foranalysis. Phosphorus NMR spectra were recorded on the two-channel BrukerAvance300 with the following instrument settings: spectrometer frequency121.498 MHz, sweep width 24,271 Hz, 64,000 data points, 30 degreeexcitation pulse, 576 transients were normally taken, each with an 8second delay time and f.i.d. acquisition time of 1.35 sec. Spectra wereprocessed with a standard exponential weighting function with 0.2 Hzline broadening before Fourier transformation.

Peaks were identified using known chemical shifts. Deacylation ofsamples with monomethylamine was also used on two samples forconfirmation of peak identity and to achieve better peak resolution.Example spectra are presented in FIG. 1. Peak area integration gaverelative molar amounts of each lipid class. Weight percent values werecalculated using molecular masses calculated from a krill sample fattyacid profile (average chain length=18.6). Total PL levels werecalculated from the PMG internal standard peak. The quantification ofthe phospholipids are shown in Table 17 for both the raw material, thefinal product and for a commercially available krill oil (Neptune KrillOil). The main polar ether lipids of the krill meal arealkylacylphosphatidylcholine (AAPC) at 7-9% of total polar lipids,lyso-alkylacylphosphatidylcholine (LAAPC) at 1% of total polar lipids(TPL) and alkylacylphosphatidyl-ethanolamine (AAPE) at <1% of TPL.

TABLE 17 Phospholipid profiles Type B krill Krill Oil obtained powderNKO in Example 7 PC 66.0 68.6 75.3 AAPC 12.0 7.0 13.0 PI 1LPC 1.2 1.30.4 PS 2LPC 7.4 13.8 2.9 LAAPC 2.2 1.2 0.9 PB 6.0 3.4 3.4 AAPE 1.5 SMGPC 1.3 DHSM NAPE 3.4 CL 5.3 2.1 LPE 0.5 LCL % PL in powder 8.3 30.047.9 or lipid sample

Analysis has been carried out on the fatty acid and ether/alcoholprofiles of the AAPC. The results are presented in Table 18.

Fatty acid profile of the alkylacylphosphatidylcholine. AAPC fatty acidAAPC alcohol composition composition alcohol % 20:5(n-3) - 46.9%; 16:047.6 20:6(n-3) - 36.1%; 18:1 17.8 18:1(n-9) - 4.6% 16:1 14.1 22:5(n-3) -2.6% 14:0 10 20:4(n-6) - 1.9% 18:0 8.6 21:5(n-3) - 1.5% 18:2 5.118:2(n-6) - 0.9% 17:0 4.4 16:1(n-9) - 0.8% 15:0-i 2.1 16:0-0.7% 15:0 1.7phytanic - 0.6% 20:1 1.4 18:3(n-3) - 0.5% 15:0-a 1.3 18:4(n-3) - 0.4%18:0-i 0.4 18:1(n-7) - 0.4% 24:1-0.4% 14:0-0.3%

The rest of alcohols (i17:0, etc.), were less than 0.3% each. Only partof 20:1 was confirmed by GC-MS. The alcohol moieties composition ofKrill AAPC was determined (identification was performed in the form of1-alkyl-2,3-diTMS glycerols on GC-MS, % of total fatty alcohols wereobtained by GC with FID). Ten other fatty acids were all below 0.3% bymass.

Example 8

The purpose of this experiment was to investigate the effect ofdifferent omega-3 fatty acid sources on metabolic parameters in theZucker rat. The Zucker rat is a widely used model of obesity and insulinresistance. Obesity is due to a mutation in the leptin receptor whichimpairs the regulation of intake. Omega-3 sources compared in this studywere fish oil (FO) and two types of krill oil. The krill oil was eitherfrom a commercial supplier (Neptune® krill oil (NKO)) or preparedaccording to Example 6 (Superba™). Four groups of rats (n=6 per group)were fed ad lib either a control diet (CTRL) or a diet supplemented witha source of omega-3 fatty acids (FO, NKO, Superba). All diets suppliedsame amount of dietary fatty acids, oleic acid, linoleic acid andlinolenic acid. Omega-3 diets (FO, NKO and Superba™) were additionallybalanced for EPA and DHA content. The Zucker rats were 4 wk old at thestart of the study with average initial weight of 250 g. At this stagethe Zucker rats can be characterized as being pre-diabetic. Rats werefed the test diets for 4 wk after which they were sacrificed and bloodand tissue samples were collected. This example shows thatsupplementation of the Zucker rat with krill oil prepared as in Example7 results in an improvement of metabolic parameters characteristic ofthe obesity induced type two diabetic condition. The effect induced bythe novel krill oil is often more pronounced than the effect of FO an inseveral cases greater than the effect induced by NKO. Specifically, theeffects of the two types of krill oil differentiated with respect to thereduction of blood LDL cholesterol levels as well as lipid accumulationin the liver and muscle (FIGS. 2-9). Furthermore, the efficacy oftransfer of DHA from the diet to the brain tissue was greatest with thekrill oil prepared as in Example 6 (FIG. 10).

Example 9

The purpose of this experiment was to investigate the effect of dietarykrill oil on metabolic parameters in high-fat fed mice and to comparethe effect of dietary krill oil with that of fish oil containing thesame amount of omega-3 fatty acids. Four groups of C57BL/6 mice (n=10per group) were fed 1) chow (N), 2) high fat diet comprising 21% butterfat and 0.15% cholesterol (HF), 3) high fat diet+krill oil (HFKO) or 4)high fat diet+fish oil (HFFO). Treatment 3 contained 2.25% (w/w) krilloil as prepared in example 5 (except that the astaxanthin content was500 ppm) which were equivalent to 0.36% omega-3 fatty acids. Treatment 4also contained 0.36% omega-3 fatty acids obtained from regular 18-12fish oil. The diets were fed to the mice for 7 weeks with free access todrinking water. Data represented in this example means+/− SE. Columnsnot sharing a common letter are significantly different (P<0.05) byANOVA followed by Tukey's multiple comparison test. N=normal chow diet(n=10); HF=high-fat diet (n=10); HFFO=high-fat diet supplemented withfish oil (n=9); HFKO=high-fat diet supplemented with krill oil (n=8).The data are presented in FIGS. 12-19.

This example shows that supplementation of high-fat fed mice with krilloil results in an amelioration of diet-induced hyperinsulinemia, insulinresistance, increase in muscle lipid content (measured as a change inmuscle mass), serum adiponectin reduction and hepatic steatosis. Thesepotentially beneficial atheroprotective effects were similar or greaterthan those achieved with a supplement containing a comparable level ofomega-3 fatty acids (see FIGS. 12-19).

Example 10

The effects of different omega-3 fatty acid sources on metabolicparameters in the Zucker rat were also investigated. The Zucker rat is awidely used model of obesity and insulin resistance. Obesity is due to amutation in the leptin receptor which impairs the regulation of intake.Omega-3 sources compared in this study were fish oil (FO) and krill oil(KO). The KO was prepared by extracting the triacylglycerides and thephospholipids from the krill meal using supercritical CO₂ with ethanolso that the final oil consisted of at 50% phospholipids, 30% omega-3fatty acids and around 1300 ppm astaxanthin. Three groups of rats (n=6per group) were fed ad lib either a control diet (CTRL) or a dietsupplemented with a source of omega-3 fatty acids (FO, KO). All dietssupplied same amount of dietary fatty acids, oleic acid, linoleic acidand linolenic acid. Omega-3 diets were additionally balanced for EPA andDHA content (see Table 19).

TABLE 19 Fatty acid content of feeds used. tot n3 totn6 n6/n3 tot UFAtot SFA UFA/SFA CTRL 0.264 2.073 7.845 4.682 2.318 2.020 TAG 0.782 2.2392.864 5.737 1.263 4.544 KO 0.807 2.230 2.764 5.291 1.709 3.096

The Zucker rats were 4 wk old at the start of the study with averageinitial weight of 250 g. At this stage the Zucker rats can benon-insulin resistant. Rats were fed the test diets for 4 wk after whichthey were sacrificed and blood and tissue samples were collected. Table20 shows the fatty acid composition of the triacylglycerides and thephospholipids for visceral adipose tissue, subcutaneous adipose tissue,liver and heart.

TABLE 20 Fatty acid composition of the VAT, SAT, Liver and Heart. TAG PLn3 18:3 n3 20:5 n3 22:5 n3 22:6 n6 18:2 n6 20:4 n3 18:3 n3 20:5 n3 22:5n3 22:6 n6 18:2 n6 20:4 VISCERAL ADIPOSE TISSUE CTRL 37.729 ^(a)  0.688^(a)  2.508 ^(a)  2.691 ^(a) 254.513 ^(a)  7.883  0.019 ^(a)  0.043 ^(a)  0.124 ^(a)   1.291   0.645 ^(a)  3.466  0.118  0.648  0.623  23.310 1.076  0.014  0.002   0.010   0.248   0.121 FO 48.558 ^(a,k) 18.667^(b) 22.605 ^(b) 29.567 ^(b) 346.205 ^(b)  8.622  0.061 ^(b)  0.096 ^(b)  0.248 ^(b)   1.379   0.320 ^(b) 10.211  3.902  5.408  7.103  68.345 1.192  0.030  0.030   0.054   0.242   0.072 KO 51.564 17.404 ^(b)23.647 ^(b) 23.783 ^(b) 385.790 ^(b)  7.282  0.111 ^(b)  0.134 ^(b)  0.373 ^(c)   1.702   0.424 ^(b)  5.586  1.723  3.275  2.930  58.334 1.240  0.014  0.009   0.045   0.456   0.067 SUBCUTANEOUS ADIPOSE TISSUECTRL 46.415 ^(a)  0.890 ^(a)  2.664 ^(a)  2.864 ^(a) 427.638 10.131 ^(a) 0.004 ^(a)  0.017   0.040   0.304   0.335  1.928  0.055  0.147  0.556 53.101  0.968  0.003  0.006   0.025   0.067   0.096 FO 59.524 20.026^(b) 16.929 ^(b) 31.099 ^(b) 473.744 10.322 ^(a)  0.035 ^(b)  0.021  0.047   0.282   0.260  2.017  1.206  0.949  2.316  19.382  0.517 0.012  0.011   0.025   0.043   0.111 KO 51.999 ^(a,k) 17.551 ^(b)16.691 ^(b) 22.806 ^(b) 496.642  7.157 ^(b)  0.021 ^(b)  0.022   0.073  0.236   0.268  1.582  1.519  3.630  1.994  64.959  0.704  0.014  0.000  0.032   0.104   0.103 LIVER CTRL 15.984  4.558 ^(a)  5.037 ^(a)  8.999^(a) 159.189 33.181 0.607  0.994 ^(a)  3.496 ^(a)  23.372 ^(a)  23.274^(a)  72.149  2.351  1.424  1.835  3.180  18.602  8.791 0.540  0.000 1.786   6.123  11.233  18.769 FO 26.911 77.719 ^(b) 28.496 ^(b) 96.935^(b) 167.123 28.139 1.494 19.770 ^(b) 14.935 ^(b)  89.729 ^(b)  57.491^(b) 115.675 12.493 50.152  8.347 63.176  34.459 14.030 0.439  0.000 7.415  59.748  27.479  79.309 KO 23.298 65.104 ^(b) 54.627 ^(b) 75.568^(b) 232.367 23.517 1.459 29.158 ^(b) 18.445 101.450 ^(b)  70.619 ^(b)133.981  5.370 26.535 30.682 30.757  58.808  8.067 0.916  0.000  6.156 46.934  16.126  58.640 HEART CTRL  9.483 ^(a)  1.590 ^(a) 23.822 ^(a)23.281 ^(a) 152.366 ^(a) 38.420 ^(a) 2.273  2.270 ^(a) 37.548 ^(a)118.815 ^(a) 302.295 ^(a) 263.031 ^(a)  1.240  0.274  8.330  4.298 27.664  4.891 0.365  0.560  5.905  24.674  25.788  12.649 FO 10.386^(a) 10.002 ^(b) 45.427 ^(b) 57.068 ^(b) 144.720 ^(a) 26.780 ^(b) 1.72918.182 ^(b) 53.216 ^(a,k) 177.093 ^(b) 380.252 ^(a,k) 181.710 ^(b) 2.179  2.814 12.818  7.927  45.675  4.804 0.278  1.580 21.323  10.530186.140   7.456 KO  6.463  8.173 ^(b) 50.499 ^(b) 48.747 ^(b) 111.583^(b) 19.826 ^(b) 2.514 35.750 ^(c) 63.534 ^(b) 252.017 ^(b) 525.519 ^(b)266.554 ^(a)  1.788  0.804 18.988  4.494  24.563  2.500 0.245  3.652 3.307  28.366 152.206  22.517

This example shows that supplementation of the Zucker rat with krill oilprepared as described above resulted in a reduction in the levels ofanandamide (AEA) and 2-arachidonoyl glycerol (2-AG) in visceral adiposetissue (FIG. 20A-B). In subcutaneous fat, the level of 2-AG were reducedcompared to fish oil and control (FIG. 21A-B). In liver and heart (FIGS.22A-B and 23A-B, respectively) the level of AEA was most efficientlyreduced with krill oil.

Furthermore, the triacylglycerol content in tissues was measured aswell. FIGS. 24 and 25 show the TAG deposition in the liver and heart,respectively. In both tissues, krill oil is the most effective inreducing ectopic fat deposition. FIG. 26 shows the cholesterol profilein rat plasma, and again krill oil is the most effective treatment. FIG.27 shows the fatty acid profile of the monocytes. Clearly, krill oil ismost effective in reducing the level of arachidonic acid and therebyreducing the inflammatory potential of the monocytes. FIG. 28 shows thelevel of TNF-alpha after lipopolysaccharide (LPS) challenge, and bothkrill and fish oils show a reduced level of TNF-alpha release comparedto the control.

Example 11

In this example, the effects on lipid metabolism, ectopic fatdeposition, and susceptibility to inflammation in Zucker fa/fa rats werestudied. Relatively low doses of dietary (n-3) LCPUFA were administeredas FO or KO. Fatty acid profiles and endocannabinoid concentrations weredetermined in different tissues to examine the possible impact of (n-3)LCPUFA on the dysregulated endocannabinoid system of Zucker rats, whichwere fed a diet containing 0.8% of energy (n-3) LCPUFA, a level lowerthan that typically used in rodent studies, to allow a more meaningfulcomparison with human studies.

Eighteen male Zucker rats (Harlan) 4 wk of age were divided into 3groups and fed for 4 wk a control diet (C) or diets supplemented witheither FO (GC Rieber Oils) or KO (Superba, Aker BioMarine). The dietswere based on the AIN-93G formulation, with substitution of soybean oilwith a blend of oils (rapeseed oil, sunflower oil, coconut oil, andlinseed oil). This allowed the 3 diets to be similar for total fattyacids and for oleic, linoleic (LA), and a-linolenic (ALA) acids. FO andKO diets were further balanced for EPA and DHA content (see Table 21).The 3 diets were prepared by Altromin GmbH & Co. KG and stored in vacuumbags to reduce (n-3) LCPUFA oxidation. The amount of 0.5 g EPA+DHA/100 gof diet, equivalent to 0.8% of energy in the rat diet, was chosen toprovide a level of (n-3) LCPUFA intake achievable in humans andcorresponds to 1.8 g/d in an 8.4-MJ/d diet in humans. All experimentswere performed according to the guidelines and protocols approved by theEuropean Union (EU Council 86/609; D. L. 27.01.1992, no. 116) and by theAnimal Research Ethics Committee of the University of Cagliari, Italy.

TABLE 21 Dietary Fatty Acid Composition. Fatty C FO KO acid g/100 g diet18:3(n-3) 0.26 0.26 0.29 18:4(n-3) 0.00 0.05 0.08 20:5(n-3) 0.00 0.290.03 22:6(n-3) 0.00 0.18 0.14 Total (n-3) 0.26 0.78 0.81 18:2(n-6) 2.072.23 2.22 20:4(n-6) 0.00 0.01 0.01 Total (n-6) 2.07 2.24 2.23(n-6):(n-3) 7.85 2.86 2.76 18:1(n-9) 2.34 2.72 2.25 Total UFA 4.68 5.745.29 12:0 1.09 0.04 0.02 14:0 0.39 0.20 0.38 16:0 0.58 0.78 1.09 18:00.22 0.21 0.19 20:0 0.03 0.04 0.03 Total SFA 2.32 1.26 1.71 UFA:SFA 2.024.54 3.10

Rats were food-deprived overnight and macrophages were isolated fromtheir peritoneal cavity. The rats were deeply anesthetized with sodiumpentobarbital (50 mg/kg intraperitoneally; Sigma-Aldrich) before beingkilled. Cells were obtained by peritoneal lavage with 60 mL of cold PBScontaining 5 mmol/L EDTA. The rats were subjected to a vigorous massageof the peritoneal area prior to collection of cells. Immediately afterdeath, blood was drawn from aorta, and liver, brain, heart, subcutaneousadipose tissues (SAT), and visceral adipose tissues (VAT) were removedand stored at 280° C.

Cells were centrifuged at 300×g; 10 min and the cell pellet was washedtwice with cold sterile PBS and suspended in DMEM, 10% heat-inactivatedfetal calf serum, penicillin (100 kU/L), and streptomycin (100 mg/L).The cell number was determined with a Coulter Counter corrected forviability determined by tryptan blue dye exclusion. The cells were thenseeded at the density of 4.0×10⁵ cells cm² and incubated for 2 h at 37°C. and 5% CO₂ atm. After removing nonadherent cells, macrophages werecultured in DMEM with 10% fetal calf serum in the presence oflipopolysaccharide (LPS) from Escherichia coli 026:B6 (Sigma Aldrich)(100 mg/L) for 24 h. The incubation time was chosen based on preliminaryexperiments that showed no substantial difference in cytokine secretionbetween 24 and 48 h. At the time indicated, supernatants and cells wereseparated and stored at 280° C. until ELISA and fatty acid analysis wereperformed. Sandwich ELISA tests were carried out all at the same time toavoid variations during the assay conditions and performed as describedby the manufacturer.

Serum C-reactive protein (Chemicon International), tumor necrosisfactor-a (TNFa), interleukin (IL)-10, and tumor growth factor-b (TGFb)(Biosource) were determined by a sandwich ELISA. Moreover, to evaluatemacrophage susceptibility to inflammatory ligands, the secretion ofTNFa, IL-6 (Bender MedSystem), IL-1b, IL-10, and TGFb were assessed inculture supernatants from peritoneal macrophages activated with LPS.Media were assayed at 800×g; 10 min to remove debris. Supernatants werefrozen at 280° C. until assayed with a sandwich ELISA (Biosource).

Total lipids were extracted from tissues using chloroform:methanol 2:1(v:v). Separation of total lipids into TAG and PL was performed aspreviously reported. Aliquots were mildly saponified as previouslydescribed to obtain FFA for HPLC analysis. Separation of fatty acids wasconducted with a Hewlett-Packard 1100 HPLC system (Hewlett-Packard)equipped with a diode array detector as previously reported. Because SFAare transparent to UV, they were measured, after methylation, by meansof a gas chromatograph (Agilent, Model 6890) equipped with split ratioof 20:1 injection port, a flame ionization detector, an autosampler(Agilent, Model 7673), a 100-m HP-88 fused capillary column (Agilent),and an Agilent ChemStation software system. The injector and detectortemperatures were set at 250° C. and 280° C., respectively. H₂ served ascarrier gas (1 mL/min) and the flame ionization detector gases were H₂(30 mL/min), N₂ (30 mL/min), and purified air (300 mL/min). Thetemperature program was as follows: initial temperature was 120° C.,programmed at 10° C./min to 210° C. and 5° C./min to 230° C., thenprogrammed at 25° C./min to 250° C. and held for 2 min.

AEA and 2-AG were measured. MAGL and FAAH activities were determined inthe heart, liver, VAT, and SAT from C and FO- and KO-supplemented rats.In particular, 2-AG hydrolysis (mostly by MAGL) was measured byincubating the 10,000×g cytosolic fraction of tissues (100 mg persample) in Tris-HCl 50 mmol/L, at pH 7.0 at 37° C. for 20 min, withsynthetic 2-arachidonoyl-[³H]-glycerol (40 Ci/mmol, ARC) properlydiluted with 2-AG (Cayman Chemicals). After incubation, the amount of[³H]-glycerol produced was measured by scintillation counting of theaqueous phase after extraction of the incubation mixture with 2 volumesof CHCl₃:MeOH 1:1 (v:v). AEA hydrolysis (by FAAH) was measured byincubating the 10,000×g membrane fraction of tissues (70 mg per sample)in Tris-HCl 50 mmol/L, at pH 9.0-10.00 at 37° C. for 30 min, withsynthetic N-arachidonoyl-[¹⁴C]-ethanolamine (110 mCi/mmol, ARC) properlydiluted with AEA (Tocris Bioscience). After incubation, the amount of[¹⁴C]-ethanolamine produced was measured by scintillation counting ofthe aqueous phase after extraction of the incubation mixture with 2volumes of CHCl₃:MeOH 1:1 (by vol.). Values in the text are means± SD.

The one-way ANOVA and the Bonferroni test for post hoc analyses wereapplied to evaluate statistical differences among groups. Wherevariances were unequal, Kruskal-Wallis non-parametric 1-way ANOVA wasused.

Growth and food intake did not differ among the 3 groups and none of therats exhibited adverse effects (data not shown). At the end of the 4-wktreatment, the body weight of the rats was 400±35 g.

In KO-supplemented rats, and to a lesser extent in the FO group, theliver TAG concentration was significantly lower than in C (FIG. 29A).The heart TAG concentration was significantly lower than C only inKO-supplemented rats (FIG. 29B).

Rats supplemented with FO or KO had 75% lower plasma LDL cholesterolconcentrations than C, whereas HDL cholesterol did not differ among thegroups (FIG. 32A). Conversely, triglyceridemia was ˜30% higher than C inboth (n-3) LCPUFA-supplemented groups (FIG. 32B).

Plasma proinflammatory (TNFa, IL-6, IL-1b) and anti-inflammatorycytokines (IL-10 and TGFb) and C-reactive protein did not differ amongthe experimental groups (see Table 22).

TABLE 22 Plasma levels of inflammatory markers in rats fed Control (C),Fish Oil (FO), or Krill Oil (KO) diets. C FO KO mg/L plasma TNFα 17.2 ±2.0 19.4 ± 3.4  19.1 ± 5.2 IL-10 10.3 ± 0.6 9.8 ± 0.9 10.9 ± 1.5 CRP382.2 ± 51.7 356.8 ± 119.8 414.7 ± 42.0 TGFβ 39.4 ± 2.1 43.9 ± 2.9  39.7± 5.0 Values are mean ± SD, n = 6. No significant differences among theexperimental groups were detected

In macrophages incubated for 24 h in the presence of LPS, TNFa secretionwas significantly lower in FO and KO rats compared with C (see Table23). Plasma IL-1b, IL-6, and IL-10 concentrations did not differ amongdietary groups following LPS stimulation.

TABLE 23 TNF-alpha release in LPS-treated peritoneal macrophages fromObese Zucker rats fed C, FO, or KO diets for four weeks. C FO KO mg/Lplasma Basal  0.6 ± 0.1  1.9 ± 0.7  0.3 ± 0.1 LPS 68.4 ± 9.4^(a) 37.2 ±9.7^(b) 42.2 ± 3.2^(b) ¹Values are means ± SD, n = 6. Means in a rowwith superscripts without a common letter differ, P < 0.05.

The VAT AEA concentration was lower in the FO and KO groups than in C(FIG. 30A), whereas 2-AG was significantly lower than C only in theKO-supplemented rats (FIG. 30B). Endocannabinoid concentrations in SATdid not differ among the 3 groups.

Liver and heart endocannabinoids were similarly affected in theKO-supplemented rats (FIG. 31A-D). AEA concentrations were ˜25% of, and2-AG concentrations ˜200% of, those of C in both tissues. In FO-fedrats, liver but not heart AEA concentrations were less than in C.

Activities of enzymes involved in endocannabinoid degradation. Changesin FAAH and MAGL activity have been observed in adipose tissues andliver of obese individuals and rodents. In this study, heart, liver,VAT, and SAT FAAH activities did not differ among the experimentalgroups (see Table 24). Conversely, MAGL activity was significantly lowerin the VAT of the FO and KO groups, and in the heart tissue of the KOgroup, compared with C (see Table 24). Liver MAGL activity tended to belower in both groups compared with C(P=0.1).

TABLE 24 FAAH and MAGL activities in the heart, liver, VAT, and SAT ofObese Zucker rats fed C, FO, or KO diets for four weeks. Tissue and FAAHMAGL diet group pmol/(min · mg tissue) Heart C 4.8 ± 1.6 2442.7 ±62.2^(a)  FO 4.1 ± 1.3 2169.8 ± 10.1^(a,b) KO 4.0 ± 1.4 2072.6 ±168.4^(b) Liver C 208.0 ± 4.5  2075.3 ± 6.6   FO 207.6 ± 4.9  1958.6 ±106.7  KO 208.9 ± 3.9  1938.7 ± 110.4  VAT C 9.7 ± 3.2  1627.5 ± 151.8*FO 7.0 ± 2.7 1257.8 ± 49.0^(b)  KO 6.2 ± 0.8 1217.4 ± 18.8^(b)  SAT C2.96 ± 1.1  1279.2 ± 8.3   FO 14.1 ± 3.8  1316.3 ± 24.7   KO 50.4 ± 25.31277.8 ± 45.1   ¹Values are means ± SD, n = 4. Within a tissue, means ina column with superscripts without a common letter differ, P < 0.05.

Plasma EPA and DHA concentrations were higher and that of ARA was lowerin the FO and KO groups compared with C (see Table 25). Interestingly,the levels of ALA and LA (only in the KO group) were higher than in Cdespite the similar levels of these fatty acids in the diets (see Table21). Peritoneal macrophages from FO- and KO-fed rats had significantlyhigher EPA and DHA and lower ARA concentrations than those from the Cgroup (see Table 26).

TABLE 25 TNF-alpha release in LPS-treated peritoneal macrophages fromrats fed Control (C), Fish Oil (FO), or Krill Oil (KO) diets. C FO KOmg/L plasma Basal   0.6 ± 0.1*   1.9 ± 0.7*   0.3 ± 0.1* LPS 68.4 ±9.4^(a) 37.2 ± 9.7^(b) 42.2 ± 3.2^(b) Values are mean ± SD, n = 6.Different letters denote significant differences (p < 0.05). *In allthree dietary groups, LPS treatment increased TNF α releasesignificantly (p < 0.05).

TABLE 26 20:5(n-3), 22:6(n-3), and 20:4(n-6) concentrations inperitoneal macrophages from rats fed Control (C), Fish Oil (FO), orKrill Oil (KO) diets (expressed as mol % of total fatty acids). C FO KOmol % 20:4(n-6) 8.9 ± 2.6^(a) 7.9 ± 1.5^(a) 5.3 ± 1.0^(b) 20:5(n-3) 0.3± 0.1^(a) 2.2 ± 0.1^(b) 1.4 ± 0.4^(b) 22:6(n-3) 0.8 ± 0.5^(a) 2.1 ±0.3^(b) 1.5 ± 0.5^(b) Values are mean ± SD, n = 6. Different lettersdenote significant differences (p < 0.05).

Dietary (n-3) LCPUFA influenced the TAG fraction of VAT and SAT withgreater incorporation of EPA, DPA, and DHA in both experimental groupscompared with C (see Table 27). The EPA and DHA levels in SAT werehigher in FO- than in KO-treated rats. ARA was significantly lower thanin C only in SAT of KO-supplemented rats; there was no difference amongthe groups in VAT. As in plasma, ALA was higher than in C in both VATand SAT of FO and KO groups. On the contrary, LA was significantlyhigher only in VAT of (n-3) LCPUFA supplemented rats compared with C. Inall 3 dietary groups, remarkable differences in fatty acid profiles ofthe PL fraction were observed, also between VAT and SAT (see Table 27).PL ARA levels of the VAT, but not SAT, were significantly less in FO-and KO-supplemented rats than in C. Levels of EPA, DPA, and DHA werehigher in the (n-3) LCPUFA-supplemented rats compared with C in VAT PL,while only EPA changed significantly in SAT PL.

TABLE 27 PUFA composition of visceral and subcutaneous adipose tissuesfrom rats fed Control (C), Fish Oil (FO), or Krill Oil (KO) diets. TAGnmol/mg lipid 18:3(n-3) 20:5(n-3) 22:5(n-3) 22:6(n-3) 18:2(n-6)20:4(n-6) VISCERAL ADIPOSE TISSUE C 37.7 ± 3.5^(a)  0.7 ± 0.1^(a)  2.5 ±0.7^(a)  2.7 ± 0.6^(a)  254.5 ± 23.3^(a) 7.9 ± 1.1 FO  48.6 ± 10.2^(a,b)18.7 ± 3.9^(b) 22.6 ± 5.4^(b) 29.6 ± 7.1^(b)  346.2 ± 68.3^(b) 8.6 ± 1.2KO 51.6 ± 5.6^(b) 17.4 ± 1.7^(b) 23.7 ± 3.3^(b) 23.8 ± 2.9^(b)  385.8 ±58.3^(b) 7.3 ± 1.2 SUBCUTANEOUS ADIPOSE TISSUE C 46.4 ± 1.3^(a)  0.9 ±0.1^(a)  2.7 ± 0.2^(a)  2.9 ± 0.6^(a) 427.6 ± 53.1 10.1 ± 1.0^(a ) FO59.5 ± 2.0^(b) 20.0 ± 1.2^(b) 16.9 ± 0.9^(b) 31.1 ± 2.3^(b) 473.7 ± 19.410.3 ± 0.5^(a ) KO 52.0 ± 1.6^(c) 17.6 ± 1.5^(c) 16.7 ± 3.6^(b) 22.8 ±2.0^(c) 496.7 ± 65.0 7.2 ± 0.7^(b) PL nmol/mg lipid 18:3(n-3) 20:5(n-3)22:5(n-3) 22:6(n-3) 18:2(n-6) 20:4(n-6) VISCERAL ADIPOSE TISSUE C 0.01 ±0.01^(a) 0.04 ± 0.01^(a )  0.1 ± 0.01^(a) 1.3 ± 0.3  0.6 ± 0.1^(a) FO0.06 ± 0.03^(b)  0.1 ± 0.03^(b)   0.3 ± 0.05^(b) 1.4 ± 0.2  0.3 ±0.1^(b) KO  0.1 ± 0.01^(c)  0.1 ± 0.01^(c)  0.4 ± 0.04^(c) 1.7 ± 0.5 0.4 ± 0.1^(b) SUBCUTANEOUS ADIPOSE TISSUE C 0.004 ± 0.003^(a) 0.02 ±0.006 0.04 ± 0.02 0.3 ± 0.1 0.3 ± 0.1 FO 0.03 ± 0.01^(b) 0.02 ± 0.01 0.05 ± 0.02  0.3 ± 0.04 0.3 ± 0.1 KO 0.02 ± 0.01^(b) 0.02 ± 0.003 0.07 ±0.03 0.2 ± 0.1 0.3 ± 0.1 Value are mean ± SD, n = 6. Different lettersdenote significant differences (p < 0.05).

TABLE 28 PUFA composition of liver and heart from rats fed Control (C),Fish Oil (FO), or Krill Oil (KO) diets. TAG PL nmol/mg lipid nmol/mglipid 18:3(n-3) 20:5(n-3) 22:5(n-3) 22:6(n-3) 18:2(n-6) 20:4(n-6)18:3(n-3) LIVER C 16.0 ± 2.3 4.6 ± 1.4a 5.0 ± 1.8a  9.0 ± 3.2a 159.2 ±18.6 33.2 ± 8.8 0.6 ± 0.54 FO  26.9 ± 12.5 77.7 ± 50.1b 28.5 ± 8.3b  96.9 ± 63.2b 167.1 ± 34.5  28.1 ± 14.0 1.5 ± 0.44 KO 23.3 ± 5.4 65.1 ±26.5b 54.6 ± 30.7b  75.6 ± 30.8b 232.4 ± 58.8 23.5 ± 8.1 1.5 ± 0.92HEART C  9.5 ± 1.2a 1.6 ± 0.3a 23.8 ± 8.3a  23.3 ± 4.3a 152.4 ± 27.7 38.4 ± 4.9a 2.3 ± 0.4  FO  10.4 ± 2.2a 10.0 ± 2.8b  45.4 ± 12.8a 57.1 ±7.9b 144.7 ± 45.7 26.78 ± 4.8b 1.7 ± 0.3  KO  6.5 ± 1.8b 8.2 ± 0.8b 50.5± 19.0b 48.7 ± 4.5b  111.6 ± 24.6b 19.82 ± 2.6b 2.5 ± 0.2  PL nmol/mglipid 20:5(n-3) 22:5(n-3) 22:6(n-3) 18:2(n-6) 20:4(n-6) LIVER C  1.0 ±0.2a  3.5 ± 1.8a 23.4 ± 6.1a  23.3 ± 11.2a 72.1 ± 18.8 FO 19.8 ± 4.5b14.9 ± 7.4b  89.7 ± 59.7a  57.5 ± 27.5b 115.68 ± 79.3  KO 29.2 ± 6.3c18.4 ± 6.2b 101.4 ± 46.9b  70.6 ± 16.1b 133.98 ± 58.6  HEART C 2.27 ±0.6a 37.5 ± 5.9a 118.8 ± 24.7a 302.3 ± 25.8  263.0 ± 12.6a FO 18.18 ±1.6b    53.2 ± 21.3a,b  177.1 ± 10.57b 380.2 ± 186.1 181.7 ± 7.5b  KO35.75 ± 3.6c  63.5 ± 3.3b 252.0 ± 28.4c 525.5 ± 152.2 266.5 ± 22.5aValue are mean ± SD, n = 6. Different letters denote significantdifferences (p < 0.05)

In liver TAG, EPA, DPA, and DHA levels were significantly elevated inthe FO and KO groups compared with C, whereas ARA levels did not differamong the groups. In liver PL, EPA and DPA concentrations were greaterin both (n-3) LCPUFA supplemented groups than in C, whereas DHA wassignificantly higher than C only in the KO group. LA was significantlygreater in the (n-3) LCPUFA-supplemented groups than in C, whereas theARA level did not differ (see Table 28). The heart TAG fatty acidprofile had higher levels of EPA, DPA, and DHA and lower levels of ARAin the FO and KO groups compared with C. LA and ALA concentrations werelower than in C only in the KO-supplemented rats. In the PL fraction ofthe (n-3) LCPUFA-supplemented groups, concentrations of EPA, DPA, andDHA were also greater than C, with higher levels in the KO group,whereas ARA was significantly less than in C only in the FO group (seeTable 28).

Example 12

Male CBA/J mice were purchased from Jackson Laboratory at six weeks ofage and were individually housed and fed 84 kcal/week of a controlAIN93M diet. At two months of age, mice were transferred to one of sixtest diets (10 mice per diet): Control, a diet supplemented with fishoil (FO), and a diet supplemented with Superba krill oil (KO). All micereceived 84 kcal/week. The supplemented diets were based onmodifications of the Control diet as described in Table 29. Amounts ofeach component are shown as grams of that component per kilogram ofdiet.

TABLE 29 Lipid and protein sources for the diets. Control Fish oil Krilloil Lipid source 40 g soybean oil 29 g soybean oil; 25 g soybean oil; 11g fish oil 15 g krill oil Protein source 140 g casein 140 g casein 140 gcasein

Body weight was measured approximately two times per month and weresimilar in all groups. At five months of age, mice were euthanized bycervical dislocation, blood was collected from the body cavity, andtissues were rapidly dissected, flash frozen in liquid nitrogen, andstored at −80° C. Gene expression profiling was performed. Total RNA wasextracted from liver tissue of seven mice per group and was processedaccording to standard protocols described by Affymetrix. Samples werehybridized on the Affymetrix Mouse Genome 430 2.0 array, which allowsfrom the detection of approximately 20,000 known genes. To determine theeffect of a test diet on the expression of a gene, the average signalintensity for the treated group was compared to the average signalintensity for that gene in the Control group. Comparisons between groupswere made using two-tailed t-tests (experimental vs. Control); a genewas considered to be significantly changed by treatment at p<0.01.

To identify functional classes of genes changed by treatment ParametricAnalysis of Gene set Enrichment (PAGE) was performed. This techniqueallows for an unbiased and highly sensitive method of detecting classesof genes that are modulated by treatment. In addition, PAGE determines az-score indicating if a gene class was activated (z-score >0) orrepressed (z-score <0) by treatment. Genes were grouped into functionalclasses using the Gene Ontology (GO) hierarchy, and GO terms that wereannotated with at least 10 but not more than 1000 genes per term wereconsidered. GO terms were considered to be significantly altered bytreatment at p<0.001. A global comparison of the GO Biological Processesmodulated by diet was made, which showed that fish oil is less bioactivethan krill oil. Moreover, in several cases (e.g., lipid biosynthesis andfatty acid metabolism), the overall effect of fish oil was in theopposite direction as observed with the KO diet.

The effect of diet on glucose metabolism was studied. PAGE analysisrevealed that the GO term “glucose metabolism” was decreased in the FOand KO diets (p=0.004-6). The ability of n-3 PUFAs to decrease glucosemetabolism compared to the control diet with the same energy intake maybe regulated at the early stages of glycolysis by decreasing glucoseuptake though the liver glucose transporter (Glut2/Slc2a2) and bydecreased phosphorylation through the liver enzyme glucokinase (Gck). KOshowed a trend for decreased Slc2a2 expression (p=0.030; see FIG. 34).It is important to note that the majority of the carbohydrate in thediets used in this study comes from sucrose (composed of glucose andfructose), and interestingly, there appears to be a shift favoringfructose metabolism in mice fed diets containing KO. As shown in FIG.34, expression of two genes involved in fructose metabolism tended to begreater in the KO group: Ketohexokinase (Khk) converts fructose tofructose-1-phosphate, and aldolase B (Aldob) convertsfructose-1-phosphate into compounds which can enter glycolysis or beused to synthesize glycogen. Taken together, the decrease in glucosemetabolism and shift towards hepatic fructose metabolism suggest thatkrill oil supplementation acts to preserve glucose for tissues such asbrain or muscle.

PAGE also revealed a trend for decreased gluconeogenesis in the KOgroup, with a nearly significant decrease in this pathway (p=0.040). Inaddition to the genes annotated in this Gene Ontology term, there areseveral well-known genes that regulate hepatic glucose production butare not annotated by the Gene Ontology consortium; interestingly, thesegenes showed a strong trend to be regulated in the KO group (see FIG.35) providing further evidence for decreased hepatic gluconeogenesis.Two of these genes (Ppargc1a and Hnf4a) are master regulators ofmetabolic gene transcription, and they have potent physiological effectson hepatic gluconeogenesis/glucose production. These genes encodeproteins that regulate metabolism by binding to DNA and enhancing theexpression of other metabolic genes in many tissues. In the liver ofhumans with type 2 diabetes and in mouse models of diabetes, theexpression of Ppargc1a and Hnf4a are increased which results increasesthe expression of genes that result in gluconeogenesis(phosphoenolpyruvate carboxykinase 1; Pck1) and aberrant glucose exportfrom the liver (glucose-6-phosphatase, G6 pc). In the current study,Ppargc1a expression was decreased in KO (p=0.014); and Hnf4a wassignificantly decreased in expression by the KO diet. There were alsomarked reductions by KO in the expression of two targets ofPpargc1a/Hnf4a (see FIG. 35), with Pck1 being significantly decreased byKO and G6 pc showing a trend for a decrease by KO (p=0.04). These datastrongly suggest that krill oil has the ability to suppress hepaticglucose production which is increased in type 2 diabetes. Suppression ofgluconeogenesis and hepatic glucose output via modulation of Ppargc1aactivity has been proposed as a strong therapeutic target for thetreatment of diabetes, provided that the intervention does not opposethe beneficial effects of Ppargc1a expression in other tissues. Thekrill supplements used in this study may functionally decrease hepaticglucose production and increase glucose uptake in tissues other thanliver.

The effect of diet on hepatic lipid metabolism was also studied. The KOdiet resulted in a gene expression profile suggesting decreased hepaticlipid accumulation. There is a significant modulation of the GO term“lipid biosynthesis.” While decreased hepatic lipid synthesis in the KOgroup may simply be a consequence of decreased substrate availability asa result of decreased glucose metabolism, this result may be of clinicalsignificance as hepatic lipid accumulation (hepatic steatosis) isassociated with insulin resistance and the metabolic syndrome in humans.Interestingly, fish oil did not significantly modulate lipidbiosynthesis in this study. Thus, it is tempting to speculate that krilloil would be a novel dietary intervention to modulate key pathways ofenergy metabolism in the liver in a manner which would oppose the effectseen in type 2 diabetes.

Although it has been reported that n-3 PUFA supplementation increasesthe expression of genes involved in fatty acid oxidation, pathwayanalysis of the gene expression data in this study showed that the GOterm “fatty acid metabolism” was significantly depressed by KO, with noeffect of FO on this pathway. This effect is underscored by the decreasein the expression of key genes involved in mitochondrial fatty acidoxidation (see FIG. 36) including the liver isoform of the rate-limitingenzyme carnitine palmitoyl transferase 1 and three enzymes involved inmitochondrial fatty acid beta oxidation (Acads, Acadm, and Acadl). Thereason for the discrepancy between this example and previous reportsshowing that n-3 PUFAs increase fatty acid oxidation is not clear.However, others have shown that expression of the Cpt1a gene isregulated by Ppargc1a, and the data from this study are in agreementwith that finding. Thus, there appears to be strong transcriptionalevidence that fatty acid metabolism is decreased in response to krilloil supplementation.

PAGE analysis also revealed that KO significantly suppressed the pathway“cholesterol biosynthesis.” Other studies have shown that krill oil hasthe ability to improve circulating triglycerides as well as cholesterollevels in rats and humans, and the current data provide molecularevidence to support those findings. Specifically, KO resulted in asignificant decrease in the expression of two key genes in the pathwayof cholesterol metabolism including the gene encoding the rate-limitingenzyme for cholesterol synthesis (Hmgcr) and the Pmvk gene which encodesa protein that catalyzes the fifth condensation reaction in cholesterolsynthesis (FIG. 37). Others have suggested that the hyperlipidemiceffects of dietary saturated fats are mediated through increasedactivity of PPARgamma coactivator, 1beta (Ppargc1b) and sterolregulatory element binding factor 2 (Srebf2). As shown in FIG. 37, weobserved a significant downregulation of both of these genes by KO.Thus, previous studies and the current study provide evidence that theliver is sensitive to the saturation of dietary fatty acids, and thatPpargc1b and Srepf2 activity may be the important regulators ofcholesterol synthesis in response to dietary fatty acid saturation.

The effect of diet on mitochondrial respiratory activity was alsostudied, as well as its implications for reduced oxidative damage. PAGErevealed that the KO diet resulted in a significant activation of the GOterm “mitochondrial respiratory chain”; this was largely caused by anincreased expression of genes encoding subunits of Complex I (NADHdehydrogenase). KO was also associated with a significant decrease inthe expression of superoxide dismutase 2 (Sod2, FIG. 34), a criticalenzyme involved in the detoxification of reactive oxygen species inmitochondria. Biochemical assays of oxidative damage (lipidperoxidation, nucleic acid oxidation) in banked tissues would reveal ifthe transcriptional effects are seen at the biochemical level. It seemsplausible, therefore, that the increased activity of the mitochondrialrespiratory chain in KO mice is related to increased mitochondrialproton leak which would result in decreased oxidative damage.

The effect of diet on inflammatory pathways was also studied, althoughno striking effects were observed. PAGE analysis revealed that there wasa trend for KO modulation of several pathways involved in inflammation,most notably an increase in the activity of “negative regulation oflymphocyte proliferation” (p<0.05), an anti-inflammatory action of KOmay be more pronounced in adipose tissue or brain.

As a result of these studies, a proposed mechanism of action for krilloil-regulation of metabolism has been developed. The data above clearlysupport a role for krill oil-supplementation to have beneficial effectson hepatic glucose and lipid metabolism. Two key regulators of thesemetabolic pathways in liver are sterol regulatory element bindingtranscription factor 1 (Srebf1) and the carbohydrate recognition elementbinding protein (MIxipl). Expression and activity of the genes encodingthese transcriptional cofactors is increased by insulin (Srebf1) andglucose (MIxipl) which results in a stimulation of glycolysis andhepatic lipogenesis leading to lipid accumulation and insulin resistancein the liver. Conversely, inhibition or deficiency of these proteinsameliorates metabolic abnormalities in mouse models of the metabolicsyndrome. The KO diet robustly decreased the expression of these twogenes (see FIG. 38), though FO had no effect. Post-translationalregulation of these transcriptional cofactors by PUFAs appears to bespecific to particularly fatty acids, although the current studysuggests that the genes Srebf1 and MIxipl are also regulated at thetranscriptional level by certain PUFAs.

In addition to the decreased expression of these transcriptionalcofactors, four genes known to be targets of these regulatory genes weredecreased in expression by a krill oil-supplemented diets (see FIG. 38).These genes include: liver pyruvate kinase (Pklr), a regulatory enzymein hepatic glycolysis; ATP citrate lyase (Acly) which catalyzes theconversion of citrate to acetyl CoA which can be used for synthesis offatty acids; fatty acid synthase (Fasn) and acetyl CoA carboxylase(Acaca) which catalyze two of the initial steps of fatty acid synthesis.Because PAGE revealed that hepatic glucose and fatty acid metabolism aresuppressed by krill oil-supplementation, the data altogether confirmthat Srebf1 and MIxipl are master regulators of hepatic metabolism, andtheir transcription and activity are modulated by krilloil-supplementation.

Example 13

This example, which was conducted using the study parameters set forthabove in Example 12, addresses the effect of fish oil and krill oil onhepatic gene expression in mice. Fish oil was much less potent inchanging the gene expression, compared to krill oil. Table 29 sets forththe pathways that are differently affected by krill oil and fish oil.

TABLE 29 Pathway GO ID Genes FC_CR FC_FO FC_KO fatty acid GO:0006631 149−6.17 1.94 −3.59 metabolic process monocarboxylic GO:0032787 202 −5.231.42 −4.08 acid metabolic process cellular lipid GO:0044255 485 −5.641.41 −5.02 metabolic process lipid metabolic GO:0006629 568 −5.49 1.50−5.18 process peroxisome GO:0005777 90 −7.23 3.59 −1.60 fatty acidGO:0019395 20 −5.49 1.95 −2.16 oxidation fatty acid GO:0009062 18 −5.412.34 −2.52 catabolic process GO ID = The list of genes within a GOpathway can be obtained at the website for the Gene Ontology Consortium.CR = calorie restriction diet.

The difference between krill oil and fish oil on the regulation of geneswithin lipid metabolism can be ascribed to their influence on keyregulators of those genes. The most important transcription factorsregulating these genes include PPAR alpha, PPAR delta, SREBP-1c,SREBP-2, ChREBP/MIxipl, HNF-4 alpha, PGC-1 alpha, and PGC-1 beta.

Krill oil led to a downregulation of all of these transcription factors,whereas fish oil had no significant effect. Also, the same pattern wasseen on the expression of target genes for each of the transcriptionfactors.

Mitochondrial genes are in general down-regulated after treatment withkrill oil. However, treatment with krill oil leads to a significantupregulation of certain classes of mitochondrial genes. Those genesappear to be associated with the electron transfer/respiratory chain,mitochondrial ribosomes, protein located at the inner mitochondrialmembrane, and ATPases.

The majority of those genes are encoded in the nucleus. However, somegenes in the respiratory chain are encoded in the mitochondria. Theunderlying mechanism behind this effect of krill oil is not known.However, it might indicate that the energy production in the liver is insome way affected. This effect is not seen after treatment with fishoil.

It will, of course, be appreciated that the above description has beengiven by way of example only and that modifications in detail may bemade within the scope of the present invention.

Throughout this application, various patents and publications have beencited. The disclosures of these patents and publications in theirentireties are hereby incorporated by reference into this application,for example, in order to more fully describe the state of the art towhich this invention pertains.

The invention is capable of considerable modification, alteration, andequivalents in form and function, as will occur to those ordinarilyskilled in the pertinent arts having the benefit of this disclosure.

While the present invention has been described for what are presentlyconsidered the preferred embodiments, the invention is not so limited.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the detailed description provided above.

What is claimed:
 1. A method for reducing risk factors for metabolicdisorders in a subject, comprising: administering to said subject aneffective amount of a krill oil composition, said krill oil compositioncomprising from about 40% w/w to about 60% w/w phospholipids, whereinsaid phospholipids comprise about 1% to 8% ether phospholipids, andabout 25% to 45% triglycerides, under conditions such that metabolicdisease risk factors of the subject are improved, wherein the metabolicdisease risk factor improvements are selected from the group consistingof a reduction in ectopic fat; an increase in the levels of EPA and DHAin the phospholipid fractions of tissues that exhibit changes inendocannabinoid concentration upon administration of said krill oilcomposition; a reduction in monoacylglyceride lipase activity in thevisceral adipose tissue; an increase in the level of plasma ALA/LA; anda decrease in the level of ARA in the subcutaneous adipose tissue. 2.The method of claim 1, wherein the metabolic disorders are selected fromthe group consisting of type II diabetes, obesity, and metabolicsyndrome.
 3. The method of claim 1, wherein said metabolic disease riskfactor improvement is a reduction in ectopic fat upon administration ofsaid krill oil composition.
 4. The method of claim 1, wherein saidmetabolic disease risk factor improvement is an increase the levels ofEPA and DHA in the phospholipid fractions of tissues that exhibitchanges in endocannabinoid concentration upon administration of saidkrill oil composition.
 5. The method of claim 1, wherein said metabolicdisease risk factor improvement is a reduction in monoglyceride lipaseactivity in the visceral adipose tissue upon administration of saidkrill oil composition.
 6. The method of claim 1, wherein said metabolicdisease risk factor improvement is an increase in the level of plasmaALA/LA upon administration of said krill oil composition.
 7. The methodof claim 1, wherein said metabolic disease risk factor improvement is adecrease in the level of ARA in the subcutaneous adipose tissue uponadministration of said krill oil composition.